Immunity (AS Biology)

An understanding of the immune system shows how cells and molecules function together to protect the body against infectious diseases
and how, after an initial infection, the body is protected from subsequent infections by the same pathogen. Phagocytosis is an immediate
non-specific part of the immune system, while the actions of lymphocytes provide effective defence against specific pathogens.

11.1 The immune system

1. describe the mode of action of phagocytes (macrophages and neutrophils)
2. explain what is meant by an antigen (see 4.1.3) and state the difference between self antigens and non-self antigens
3. describe the sequence of events that occurs during a primary immune response with reference to the roles of:
macrophages
B-lymphocytes, including plasma cells
T-lymphocytes, limited to T-helper cells and T-killer cells
4. explain the role of memory cells in the secondary immune response and in long-term immunity

Key words:

immune system: the body’s internal defence system

antigen: a substance that is foreign to the body and stimulates an immune response (e.g. any large molecule such as a protein)
self: refers to substances produced by the body that the immune system does not recognise as foreign, so they do not stimulate an
immune response
non-self: refers to any substance or cell that is recognised by the immune system as being foreign and will stimulate an immune
response
antibody: a glycoprotein (immunoglobulin) made by specialised lymphocytes in response to the presence of a specific antigen; each
type of antibody molecule has a shape that is complementary to its specific antigen
immune response: the complex series of responses of the body to the entry of a foreign antigen; it involves the activity of
lymphocytes and phagocytes
clonal selection: individual lymphocytes have cell surface receptors specific to one antigen; this specificity is determined as
lymphocytes mature and before any antigens enter the body (during an immune response the only lymphocytes to respond are those
with receptors specific to antigens on the surface of the invading pathogen)
clonal expansion: the increase in number of specific clones of lymphocytes by mitosis during an immune response
plasma cell: short-lived, activated B-lymphocyte produced during clonal expansion; plasma cells produce and release antibody
molecules
memory B cell: long-lived, activated B-lymphocyte that is specific to one antigen; memory cells are activated to differentiate
(develop) into plasma cells during secondary immune responses to the specific antigen
primary immune response: the first immune response to a specific antigen
secondary immune response: the second and any subsequent immune responses to a specific antigen
immunological memory: the ability of the immune system to mount a larger and more rapid response to an antigen that has already
been encountered before
variable region: region of an antibody molecule composed of parts of the light and heavy polypeptide chains that form the antigen-
binding site; the amino acid sequences of the variable site form a specific shape that is complementary to a particular antigen
antigen presentation: the process of preparing antigens and exposing them on the surface of host cells (e.g. macrophages) for
recognition by T-lymphocytes
T-helper cell: type of T-lymphocyte that secretes cytokines to coordinate activity during immune responses
T-killer cell: type of T-lymphocyte that attaches to cells, releasing toxic substances to kill infected cells and cancer cells
cytokine: any signalling molecule released by cells to influence the growth and/or differentiation of the same or another cell

Introduction to immunity

Immunity = Protection against diseases

Immune system = The body's defence system

Lines of defences against diseases:

 To prevent infectious diseases from entering and spreading

1. First line of defence

External, non-specific
2. Second line of defence
Internal, non-specific immune response
Involves phagocytes
3. Third line of defence
Internal, specific immune response
Involves lymphocytes

Both non-specific and specific defences work together to protect the body against diseases.

First lines of defence:

Physical barriers:

1. Skin
2. Mucosa (Mucosa is the moist, inner lining of some organs and body cavities such as the nose, mouth, lungs, and stomach. Glands in
the mucosa make mucus (a thick, slippery fluid). Also called mucous membrane) of the...
Respiratory system
Gastrointestinal tract
Urinary tract

Chemical barriers:

1. HCl in stomach
2. Lysozyme in sweat and tears
3. Lactic acid in vagina

Immune response = the body's immune reaction towards non-self antigens

Involves WBCs that are made in bone marrow:

1. Phagocytes (mostly non-specific defence)

Neutrophils
Monocytes
→ Which mature into macrophages
2. Lymphocytes (mostly specific defence)
B-lymphocytes
T-lymphocytes

Chapter 8 Recap:

Phagocytes:

Produced throughout life

Function of phagocytes:

Patrol in blood, tissues and organs

Remove dead cells and pathogens
→ By phagocytosis Cell membranes and transport (AS Biology) > Endocytosis
Phagocytes are involved in non-specific defense
→ Responds to many different non-self antigens
Appearance of phagocytes:

Lobed nuclei
Granular cytoplasm – due to many vesicles

Neutrophils:

Has multi-lobed nucleus

Have receptor proteins on its membrane
→ To identify pathogens as non-self
When there is an infection, large numbers of neutrophils are released from bone marrow
→Accumulate at site of infection
Short-lived (few hours-day)
→ Dies after pathogens
Dead neutrophils form pus

Monocytes:

Monocytes mature into macrophages

Have lobed nucleus / kidney-bean shaped
Are larger than neutrophils
Have receptor proteins on its membrane
→ To identify pathogens as non-self
Monocytes circulate in blood
→ They mature into macrophages when it leaves blood and enter organs
Are long-lived cells
Macrophages found in organs such as liver, lungs, spleen, kidney, lymph nodes

Lymphocytes:

Produced in bone marrow before birth

Function of lymphocytes:

Involved in specific immune responses

→ Responds to only specific non-self antigens
Mature lymphocytes circulate in the blood and lymph
→ Accumulate at sites of infection

Appearance of lymphocytes:

Smaller than phagocytes

Has larger round nucleus
Has little cytoplasm

2 main types of lymphocytes:

Both made in bone marrow, but mature in different places and have different functions.

1. B-lymphocytes (B cells)
Mature in bone marrow
Produces antibodies
2. T-lymphocytes (T cells)
Mature in thymus
Does NOT produce antibodies

Initial immune response:

Innate immune system provides an immediate response to infection.

The first to respond are the macrophages. They recognise by specific characteristics that occur on pathogens but do not occur on the cells
of the body. These characteristics are called pathogen associated molecular patterns or PAMPs. They recognise PAMPs using various
receptor types including toll-like receptors.

Link to more information regarding innate immune system, complement immune system and specific innate immune system: innate immune
system, complement immune system and specific immune system

Antigens
Antigens

2 types: Self and Non-self

In general, antigens are macromolecules on cell surfaces
E.g. protein, glycoprotein, glycolipid, polysaccharides, etc.

1. Non-self antigens = macromolecules that activates an immune response

Macromolecules are found on...
foreign materials' surface (e.g. pathogen, allergen)
surface membrane of infected host cells
→ Stimulates production of antibodies

2. Self antigens / cell marker

Macromolecules on cell surface membranes of host cells
These cell surface antigens do NOT trigger body's immune system
No antibodies are produced

Note: when we say antigen in general, we are usually referring to NON-self antigen

Antigens, self & non-self

 Every cell in the human body has markers that identify it
Microorganisms (both pathogenic and non-pathogenic), such as bacteria and viruses, also have their own unique markers
These markers are called antigens (which are macromolecules) and they allow cell-to-cell recognition
Antigens are found on cell surface membranes, bacterial cell walls, or the surfaces of viruses
Some glycolipids and glycoproteins on the outer surface of cell surface membranes act as antigens
Antigens can be either self antigens or non-self antigens:
Antigens produced by the organism's own body cells (those that the immune system does not recognise as foreign antigens) are
known as self antigens
Self antigens do not stimulate an immune response
Antigens not produced by the organism’s own body cells (those that the immune system recognises as being foreign eg. the
antigens found on pathogenic bacteria and viruses or if a person receives a different blood type during a transfusion) are known as
non-self antigens
Non-self antigens stimulate an immune response

Phagocytes: Origin & mode of action

Phagocytes: Origin & Mode of Action

Phagocytes are white blood cells that are produced continuously in the bone marrow
They are stored in the bone marrow before being distributed around the body in the blood
They are responsible for removing dead cells and invasive microorganisms
They carry out what is known as a non-specific immune response
There are two main types of phagocyte, each with a specific mode of action. The two types are:
Neutrophils
Macrophages
As both are phagocytes, both carry out phagocytosis (the process of recognising and engulfing a pathogen) but the process is slightly
different for each type of phagocyte

Neutrophils

Neutrophils

Neutrophils travel throughout the body and often leave the blood by squeezing through capillary walls to ʻpatrolʼ the body tissues
During an infection, they are released in large numbers from their stores
However, they are short-lived cells
Mode of action:
Chemicals released by pathogens, as well as chemicals released by the body cells under attack (eg. histamine), attract
neutrophils to the site where the pathogens are located (this response to chemical stimuli is known as chemotaxis)
Neutrophils move towards pathogens (which may be covered in antibodies)
The antibodies are another trigger to stimulate neutrophils to attack the pathogens (neutrophils have receptor proteins on their
surfaces that recognise antibody molecules and attach to them)
Once attached to a pathogen, the cell surface membrane of a neutrophil extends out and around the pathogen, engulfing it and
trapping the pathogen within a phagocytic vacuole
This part of the process is known as endocytosis
The neutrophil then secretes digestive enzymes into the vacuole (the enzymes are released from lysosomes which fuse with the
phagocytic vacuole)
These digestive enzymes destroy the pathogen
After killing and digesting the pathogens, the neutrophils die
Pus is a sign of dead neutrophils

The stages of phagocytosis, as carried out by a neutrophil:

Macrophages

Role of macrophages

Initiates / starts the immune response

Mechanism:

Has various receptor proteins on cell surface

Can detect non-self antigens
Non-specific
Engulf pathogen / foreign material via phagocytosis (Link to Topic 4: Cell membranes and transport (AS Biology) > Endocytosis)
Fusion of phagocytic vacuole with lysosome
Cuts up pathogen using lysozymes
Antigens (of pathogen) presented on its cell surface
→ Macrophages act as antigen-presenting cells (APC)
Some cell fragments released by exocytosis
APCs can activate / stimulate lymphocytes

Note: Other APCs include B cells and other types of phagocytes too!
Macrophages

Macrophages are larger than neutrophils and are long-lived cells

Rather than remaining in the blood, they move into organs including the lungs, liver, spleen, kidney and lymph nodes
After being produced in the bone marrow, macrophages travel in the blood as monocytes, which then develop into macrophages once
they leave the blood to settle in the various organs listed above
Mode of action:
Macrophages play a very important role in initiating an immune response
Although they still carry out phagocytosis in a similar way to neutrophils, they do not destroy pathogens completely
They cut the pathogens up so that they can display the antigens of the pathogens on their surface (through a structure called the
major histocompatibility complex)
These displayed antigens (the cell is now called an antigen-presenting cell) can then be recognised by lymphocytes (another type
of white blood cell)

The vacuole formed around a bacterium once it has been engulfed by a phagocyte is called a phagosome. A lysosome fuses with the
membrane of the phagosome (to form a phagolysosome) and releases lysozymes (digestive enzymes) to digest the pathogen.

Primary immune response

Primary Immune Response

Lymphocytes are another type of white blood cell

They play an important part in the specific immune response
They are smaller than phagocytes
They have a large nucleus that fills most of the cell
They are produced in the bone marrow before birth
There are two types of lymphocytes (with different modes of action). The two types of lymphocytes are:
B-lymphocytes (B cells)
T-lymphocytes (T cells)

Maturation and activation of B-lymphocytes

Lymphocytes

Millions of different types of B and T-lymphocytes with receptors of different shapes

SPECIFIC = each type of lymphocyte responds to 1 type of antigen only

E.g. Each type of B cell produces 1 type of antibody receptor

→ which responds to 1 type of antigen only

So the body can respond to almost any type of pathogen

Only mature lymphocytes can circulate in the blood & lymph and carry out immune responses.
Maturation of B-lymphocytes:

1. All B cells are formed in the bone marrow before birth

→ Genes in B cells that code for antibodies code for different types of antibodies for different types of B cells
2. Forms a specific antibody that acts as glycoprotein receptor on surface membrane of B cells
→ Binds to specific antigen that is complementary in shape
3. B-lymphocytes divides and mature in bone marrow
→ Mature B-lymphocytes circulate in blood and concentrate in liver, spleen and lymph nodes

Antibodies

A.k.a immunoglobins
Globular glycoproteins (carbo part not shown in diagram)
Made of 4 polypeptide chains:
2 heavy chains
2 light chains
→ Quaternary structure
Held together by disulfide bonds
→ Gives stability

Three regions on antibody:

1. Variable region (Fab)

Formed by light and heavy chains
Provide 2 identical antigen-binding sites
Specific for binding antigen
→ Complementary shape to antigen
→ Shape determined by primary structure = specific sequence of amino acids
R groups at antigen-binding sites forms hydrogen bonds and ionic bonds with specific antigen

Sequence of amino acids at the variable region is different for each type of antibody

→ Each type of antibody binds to different antigens

 2. Constant region (Fc)
Formed by light and heavy chains
When circulating in blood: binds to receptors on phagocytes
When antibody acts as B cell receptor: attach to cell surface membrane of B cell
Gives antibody class

Antibody classes:

3. Hinge region
Held by disulfide bridges
Gives flexibility when binding to antigen

Action of antibodies:
Action of B-lymphocytes:

1. Pathogens invade
2. Antigen-presenting cell formation
3. Only specific B-lymphocytes that has receptors with the complementary shape to antigen will be activated
→ Clonal selection
4. B cells divide by mitosis
Clonal expansion
5. Activated B cells can then develop into plasma cells and memory cells

Plasma cells

Short-lived (few weeks)

Produce and secrete antibodies rapidly
→ by exocytosis
→ into blood plasma, lymph, lungs and stomach lining
Antibodies are glycoproteins
So plasma cells have extensive network of RER and Golgi
Memory cells

Long-lived, remain in circulation

Provides long term immunity
Lasts for many years/lifetime
Enables faster response during 2nd invasion of same antigen, as many memory cells are circulating
During 2nd invasion, it divides rapidly (clonal expansion)
→ Form more plasma cells → more antibodies
→ Infection is destroyed before symptoms develop

When the antigen enters the body for the first time, the small numbers of B cells with cell membrane receptors complementary to the
antigen are stimulated to divide by mitosis. This stage is known as clonal selection. The small clone of cells with receptors specific to
antigens on the surface of the invading pathogen divides repeatedly by mitosis in the clonal expansion stage so that huge numbers of
identical B cells are produced over a few weeks.

Some of the activated B cells become plasma cells that produce antibody molecules very quickly – up to several thousand a second. Plasma
cells secrete antibodies into the blood, lymph or onto the linings of the lungs and the gut. These plasma cells do not live long: after
several weeks their numbers decrease. The antibody molecules they have secreted stay in the blood for longer, however, until they too
eventually decrease in concentration.

Other B cells become memory cells. These cells remain circulating in the body for a long time. If the same antigen is reintroduced a few
weeks or months after the first infection, memory cells divide rapidly and develop into plasma cells and more memory cells. This is
repeated on every subsequent invasion by the pathogen with the same antigen, meaning that the invading pathogens can be destroyed and
removed before the development of any symptoms of the disease.

Summary of action by B-lymphocytes:

B-lymphocytes

B-lymphocytes (B cells) remain in the bone marrow until they are mature and then spread through the body, concentrating in lymph
nodes and the spleen
Millions of types of B-lymphocyte cells are produced within us because as they mature, the genes coding for antibodies are changed to
code for different antibodies
Once mature, each type of B-lymphocyte cell can make one type of antibody molecule
At this stage, the antibody molecules do not leave the B-lymphocyte cell but remain in the cell surface membrane
Part of each antibody molecule forms a glycoprotein receptor that can combine specifically with one type of antigen

Origin and maturation of B-lymphocytes:

 When an antigen enters the body for the first time, the small numbers of B-lymphocytes with receptors complementary to that antigen
are stimulated to divide by mitosis
This is known as clonal selection
As these clones divide repeatedly by mitosis (the clonal expansion stage) the result is large numbers of identical B-lymphocytes being
produced over a few weeks
During an immune response, these B-lymphocytes then form two types of cell:
Some of these B-lymphocytes become plasma cells that secrete lots of antibody molecules (specific to the antigen) into the blood,
lymph or linings of the lungs and the gut
These plasma cells are short-lived (their numbers drop off after several weeks) but the antibodies they have secreted stay in the
blood for a longer time
The other B-lymphocytes become memory cells that remain circulating in the blood for a long time
This response to a newly encountered antigen is relatively slow and is known as a primary immune response

During a primary immune response, B-lymphocytes form two types of cell:

The changes in antibody concentration in the blood during a primary and secondary response to the same antigen:
Maturation and activation of T-lymphocytes

Maturation of T-lymphocytes:

1. All T cells produced in bone marrow before birth.

2. Maturation in thymus gland
→ Thymus shrinks after puberty
Produce specific T cell receptors on cell surface membrane
→ Binds to specific antigen that is complementary in shape
→ T cell receptor's structure similar to antibodies
3. Mature T cells circulate in blood and lymph

Action of T-lymphocytes:

1. Pathogens invade
2. Antigen presenting cell formation
3. Only specific T-lymphocytes that has receptors with the complementary shape to antigen will be activated
→ Clonal selection
4. T cell divides by mitosis
→ Clonal expansion
5. Activated T cells develop into T helper cells and T killer cells
T Helper Cells
Functions:

1. Secrete cytokines / interleukins which...

a. Stimulate specific B cells
To divide and develop into plasma cells and memory B cells
Increased antibody levels
b. Stimulate macrophages
To carry out phagocytosis more vigorously
c. Stimulate killer T cells
To divide and produce more toxins
2. Form T helper memory cells
Secondary response
Long term immunity

Cytotoxic T Killer Cells

Functions:

1. Seeks out infected host cells (including APC, cancer cells) and pathogens and destroys them
a. Attach to surface of cells
b. 'Punch' holes into cells
c. Secrete toxins into cells
E.g. hydrogen peroxide, perforin

2. Forms killer T memory cells

Secondary responses
Long term immunity as it is long-lived

A summary of the immune response:

T-lymphocytes

Immature T-lymphocytes leave the bone marrow to mature in the thymus

Mature T-lymphocytes have specific cell surface receptors called T cell receptors
These receptors have a similar structure to antibodies and are each specific to one antigen

Mature T cells have specific cell surface receptors called T-cell receptors. T-cell receptors have a structure similar to that of antibodies,
and they are each specific to one antigen. T cells are activated when they recognise this antigen on another cell of the host (that is, on
the person’s own cells). Sometimes this cell is a macrophage that has engulfed a pathogen and cut it up to expose the pathogen’s surface
molecules, or it may be a body cell that has been invaded by a pathogen and is similarly displaying the antigen on its cell surface
membrane as a kind of ‘help’ signal. The display of antigens on the surface of cells in this way is known as antigen presentation. The T
cells that have receptors complementary to the antigen respond by dividing by mitosis to increase the number of cells. T cells go through
the same stages of clonal selection and clonal expansion as clones of B cells.

There are two main types of T cell:

T-helper cells
T-killer cells (also known as T-cytotoxic cells).

When T-helper cells are activated, they release cytokines – cell-signalling molecules that stimulate appropriate B cells to divide, develop
into plasma cells and secrete antibodies. Some T-helper cells secrete cytokines that stimulate macrophages to carry out phagocytosis more
vigorously. T-killer cells search the body for cells that have become invaded by pathogens and are displaying foreign antigens from the
pathogens on their cell surface membranes. T-killer cells recognise the antigens, attach themselves to the surface of infected cells, and
secrete toxic substances such as hydrogen peroxide, killing the body cells and the pathogens inside. Some T-helper cells secrete cytokines
that stimulate T-killer cells to divide by mitosis and to differentiate by producing vacuoles full of toxins.

Memory T-helper cells and memory T-killer cells are produced, which remain in the body and become active very quickly during the
secondary response to antigens.

Origin and maturation of T-lymphocytes:

 T-lymphocytes are activated when they encounter (and bind to) their specific antigen that is being presented by one of the hostʼs
cells (host cells being the humanʼs own cells)
This antigen-presenting host cell might be a macrophage or a body cell that has been invaded by a pathogen and is displaying the
antigen on its cell surface membrane
These activated T-lymphocytes (those that have receptors specific to the antigen) divide by mitosis to increase in number (similar to
the clonal selection and clonal expansion of B-lymphocytes)

These T-lymphocytes differentiate into two main types of T cell:

helper T cells
killer T cells

Helper T cells release cytokines (hormone-like signals) that stimulate B-lymphocytes to divide and develop into antibody-secreting plasma
cells. Some helper T cells secrete cytokines that stimulate macrophages to increase their rates of phagocytosis. Some helper T cells also
stimulate killer T cells to divide and produce more toxins.

Killer T cells attach to the antigens on the cell surface membranes of infected cells and secrete toxic substances that kill the body cells,
along with the pathogen inside.

The functions of T-lymphocytes during an immune response:

Memory cells & immunity
Memory Cells & Long-Term Immunity
 During an immune response, B-lymphocytes form two types of cell: plasma cells and memory cells
Memory cells form the basis of immunological memory – the cells can last for many years and often a lifetime
There are two types of immune response:
Primary immune response (responding to a newly encountered antigen)
Secondary immune response (responding to a previously encountered antigen)

Primary immune response

When an antigen enters the body for the first time, the small numbers of B-lymphocytes with receptors complementary to that antigen
are stimulated to divide by mitosis
This is known as clonal selection
As these clones divide repeatedly by mitosis (the clonal expansion stage) the result is large numbers of identical B-lymphocytes being
produced over a few weeks
Some of these B-lymphocytes become plasma cells that secrete lots of antibody molecules (specific to the antigen) into the blood,
lymph or linings of the lungs and the gut
These plasma cells are short-lived (their numbers drop off after several weeks) but the antibodies they have secreted stay in the
blood for a longer time
The other B-lymphocytes become memory cells that remain circulating in the blood for a long time
This response to a newly encountered pathogen is relatively slow

Secondary immune response

If the same antigen is found in the body a second time, the memory cells recognise the antigen, divide very quickly and differentiate
into plasma cells (to produce antibodies) and more memory cells
This response is very quick, meaning that the infection can be destroyed and removed before the pathogen population increases too
much and symptoms of the disease develop
This response to a previously encountered pathogen is, relative to the primary immune response, extremely fast
 T-lymphocytes also play a part in the secondary immune response
They differentiate into memory cells, producing two main types:
Memory helper T cells
Memory killer T cells
Just like the memory cells formed from B-lymphocytes, these memory T cells remain in the body for a long time
If the same antigen is found in the body a second time, these memory T cells become active very quickly

Immunological memory (made possible by memory cells) is the reason why catching certain diseases twice is so unlikely. For example,
there is only one strain of the virus that causes measles, and each time someone is reinfected with this virus, there is a very fast
secondary immune response so they do not get [Link], some infections such as the common cold and influenza are caused by viruses
that are constantly developing into new strains. As each strain has different antigens, the primary immune response (during which we
often become ill) must be carried out each time before immunity can be achieved.

Classification of blood cells

images for classification of blood cells:
 Features Neutrophils Macrophages

Definition Neutrophils are the most abundant type of white blood cells, and Macrophages are a large specialised type of white blood
the first to travel to the site of an infection cells that are capable of presenting antigens

Size Smaller Larger

Lifespan Short Long

Dominance at the Dominate the infected site early Dominate infected sites at larger stages (1 to 2 days after
infected site infection)

Antigen presenting Not antigen presenting cells Antigen presenting cells

cells

Nucleus Polymorphonuclear as the nucleus has several lobes Nucleus is horseshoe-shaped

Granules Granulocytes as they have a lot of granules Agranulocytes as they do not have granules

Rejection
Immunological basis of graft rejection and mechanism of graft rejection:
Question on graft rejection:

Question on blood groups and if they will be accepted:

11.2 Antibodies and vaccination

1. relate the molecular structure of antibodies to their functions
2. outline the hybridoma method for the production of monoclonal antibodies
3. outline the principles of using monoclonal antibodies in the diagnosis of disease and in the treatment of disease
4. describe the differences between active immunity and passive immunity and between natural immunity and artificial immunity
5. explain that vaccines contain antigens that stimulate immune responses to provide long-term immunity
 6. explain how vaccination programmes can help to control the spread of infectious diseases

Key words:

active immunity: immunity gained when an antigen enters the body, an immune response occurs and antibodies are produced by plasma
cells
natural active immunity: immunity gained by being infected by a pathogen
vaccine: a preparation containing antigens to stimulate active immunity against one or several diseases
artificial active immunity: immunity gained by putting antigens into the body, either by injection or by mouth
vaccination: giving a vaccine containing antigens for a disease, either by injection or by mouth; vaccination confers artificial active
immunity without the development of symptoms of the disease
passive immunity: the temporary immunity gained without there being an immune response
artificial passive immunity: the immunity gained by injecting antibodies
natural passive immunity: the immunity gained by a fetus when maternal antibodies cross the placenta or the immunity gained by an
infant from breast milk
herd immunity: vaccinating a large proportion of the population; provides protection for those not immunised as transmission of a
pathogen is reduced
ring immunity: vaccinating all those people in contact with a person infected with a specific disease to prevent transmission in the
immediate area
monoclonal antibody (Mab): an antibody made by a single clone of hybridoma cells; all the antibody molecules made by the clone have
identical variable regions so are specific to one antigen
hybridoma: a cell formed by the fusion of a plasma cell and a cancer cell; it can both secrete antibodies and divide to form other cells
like itself

Antibodies: Structure & functions

Structure

Antibodies are globular glycoproteins called immunoglobulins

Antibodies have a quaternary structure (which is represented as Y-shaped), with two ‘heavy’ (long) polypeptide chains bonded by
disulfide bonds to two ‘light’ (short) polypeptide chains
Each polypeptide chain has a constant region and variable region
The constant regions do not vary within a class (isotype) of antibodies but do vary between the classes. The constant region
determines the mechanism used to destroy the antigens
There are 5 classes of mammalian antibodies each with different roles
The amino acid sequence in the variable regions of the antibodies (the tips of the "Y") are different for each antibody. The variable
region is where the antibody attaches to the antigen to form an antigen-antibody complex
At the end of the variable region is a site called the antigen-binding site. Each antigen-binding site is generally composed of 110 to 130
amino acids and includes both the ends of the light and heavy chains
The antigen-binding sites vary greatly giving the antibody its specificity for binding to antigens. The sites are specific to the epitope
(the part of the antigen that binds to the antibody)
A pathogen or virus may therefore present multiple antigens, so different antibodies need to be produced
The ʻhingeʼ region (where the disulfide bonds join the heavy chains) gives flexibility to the antibody molecule which allows the
antigen-binding site to be placed at different angles when binding to antigens
This region is not present in all classes of antibodies

A model of the generalised structure of an antibody molecule:

Each antibody will have a different variable region with an antigen-binding site that matches one antigen or toxin produced by a pathogen.
The antigen-binding site (and therefore the antibody) is specific to one antigen.

Function

Antibodies are produced by B-lymphocytes

Antibodies bind to specific antigens that trigger the specific immune response. Every antigen has one antibody
Antigens include pathogens and their toxins, pollen, blood cell surface molecules and the surface proteins found on transplanted tissues
Antibodies are divided into five major classes (isotypes), each with a different role
The function of antibodies differ:
Antibodies can combine with viruses and toxins of pathogens (e.g. bacteria) to block them from entering or damaging cells
Antibodies can act as anti-toxins by binding to toxins produced by pathogens (e.g. the bacteria that cause diphtheria and tetanus)
which neutralises them making them harmless
Antibodies can attach to bacteria making them readily identifiable to phagocytes, this is called opsonisation. Once identified, the
phagocyte has receptor proteins for the heavy polypeptide chains of the antibodies, which enables phagocytosis to occur
Antibodies can attach to the flagella of bacteria making them less active, which makes it easier for phagocytes to do phagocytosis
Antibodies act as agglutinins causing pathogens carrying antigen-antibody complexes to clump together (agglutination). This
reduces the chance that the pathogens will spread through the body and makes it possible for phagocytes to engulf a number of
pathogens at one time
Antibodies (together with other molecules) can create holes in the cell walls of pathogens causing them to burst (lysis) when
water is absorbed by osmosis

Functions of antibodies:
Here in immunity, pathogens can also mean cancer cells as well as own cells (those that are affected by autoimmune diseases).

Antigens are proteins from anywhere, not just those found on pathogens. (including those on cancer cells, own cells, toxins produced by
pathogens, etc.)

Functions of antibodies:

1. Attach to antigens → Form antigen-antibody complex

Leads to destruction of pathogens
2. Attach to toxins (antigens) → Neutralise toxins
3. Attach to receptors (on pathogens) → Disrupt the function of receptors
4. Attach to pathogens → Clump together → "Agglutination"
5. Act as opsonins → Present to phagocytes
Antibodies attach to pathogens → makes it easier for phagocytes to recognise and destroy those pathogens
6. Antibody dependent cell mediated cytotoxicity

Monoclonal antibodies
Monoclonal = only 1 type of antibody, specific for 1 antigen

Making monoclonal antibodies: The hybridoma method

Problem (regarding manufacturing antibodies in the lab):

B cells that divide by mitosis DO NOT produce antibodies

Plasma cells that secrete antibodies DO NOT divide

Solution:
Fuse plasma cells + cancer/myeloma cells

→ Hybridoma cells that CAN divide and CAN produce antibodies

How to produce?

1. Inject foreign antigen (e.g. pathogen) into mice

2. Allow time for immune response to occur
3. Collect plasma cells from spleen
4. Fuse plasma cells with cancer cells to produce hybridoma cells
→ Use fusogen for fusion
5. Clone hybridoma cells
→ Use HAT medium for hybridoma growth
6. Screen for cell secreting desired antibody
→ By separating cells and cultures in individual wells
→ Select only one type
7. Grow hybridoma cells in large scale culture
The Hybridoma Method

Monoclonal antibodies are artificially produced antibodies produced from a single B cell clone
The hybridoma method is a method used to make monoclonal antibodies (Mabs)
The method enables large quantities of identical antibodies to be produced
The hybridoma method solved the problem of having B cells that could divide by mitosis but not produce antibodies and plasma cells
that could produce antibodies but not divide
This method was established in the 1970s
Monoclonal antibodies bind antigens, in the same way naturally produced antibodies do
They are produced by injecting mice with an antigen that stimulates the production of antibody-producing plasma cells
Isolated plasma cells from the mice are fused with immortal tumour cells, which result in hybridoma cells
These hybrid cells are grown in a selective growth medium and screened for the production of the desired antibody
They are then cultured to produce large numbers of monoclonal antibodies
Monoclonal antibodies have multiple applications to include diagnostics, treating disease, food safety testing and pregnancy testing

Uses of monoclonal antibodies

Usage of Mabs:

1. Diagnosis
Monoclonal antibodies have same specificity and detects only one antigen
→ Can distinguish between different pathogens / strains
→ Fast diagnosis than having to culture pathogen
→ Less labour intensive
→ Quicker diagnosis = quicker treatment
Can be tagged with a fluorescent label/dye
 → Can detect location of tissues expressing antigen
→ E.g. cancer cells, blood clots, etc.
Cheap, safe, fast results, easy to use, accurate
Other than diagnosis, monoclonal antibodies are also used in blood typing, pregnancy tests, etc.
2. Treatment
Used to target specific diseased cell by binding to receptors on its cell surface
→ Can kill cell by stimulating the immune system
→ Can attach radioactive substance / drug to Mabs to kill cell
Can bind to antigens on pathogens
→ Result in artificial passive immunity

Problems of using mabs in treatment

Problems:

Cause some side effects

Antibodies made in animals recognised as non-self
Trigger immune response in humans
→ Allergic reaction
Remains in the body for short period of time as it is destroyed
Needs to administered more than once in small amounts

Solution: Humanise Mabs

1. Alter genes that code for heavy and light chains of antibodies
→ Code for human antibodies instead of mice and rabbit's
2. Changing type and position of sugar groups attached to heavy chains
→ Arrangement of sugar groups same as human antibodies

Diagnostic uses of monoclonal antibodies

Monoclonal antibodies can be used diagnostically for:

Pregnancy tests
Diagnosing HIV
Detecting the presence of pathogens such as Streptococcus bacteria
Distinguishing between Herpes I and Herpes II
Blood typing before transfusions and tissue typing before transplants
Detecting the presence of antibiotics in milk
Detecting cancer cells
Monoclonal antibodies can also be used to locate the position of blood clots for patients thought to have deep vein thrombosis. This
occurs by:
Injecting a mouse with human fibrin (the main protein found in blood clots)
These cells are collected from the mouse spleen
The plasma cells are then fused with tumour cells forming hybridomas that produce antifibrin antibodies
To detect where the antibodies are binding to fibrin molecules, a radioactive chemical (producing gamma radiation) is attached to
the antibodies making them radioactively labelled
 A gamma-ray camera is used to detect where these radioactively labelled antibodies have attached to a fibrin molecule, hence
indicating where blood clots can be found
Generally monoclonal antibodies are used only once

Diagnostic use of monoclonal antibodies - test for HIV:

Therapeutic uses of monoclonal antibodies

Therapeutically monoclonal antibodies have multiple applications to include:

Treatment for the rabies virus, (which can be potentially fatal), by injecting purified antibodies
The prevention of transplanted organ rejection, achieved by intervening with the T cells involved in the rejection process
Autoimmune therapies for allergic asthma and rheumatoid arthritis; here monoclonal antibodies are able to bind and deactivate
factors involved in the inflammatory response
Treatment for diseases caused by the overproduction or inappropriate production of B-cells (eg. leukaemia, multiple sclerosis and
myasthenia gravis); the antibody (rituximab) binds to cell surface receptor proteins on B-cells (not plasma cells) and causes the
death of the cells
Prevention of blood clotting following angioplasty procedures; here monoclonal antibodies bind to receptors on the platelet surface
thereby inhibiting fibrinogen from binding and subsequent clotting from ensuing
Targeted treatment of breast cancer; Herceptin (trastuzumab) is a monoclonal antibody used to treat breast cancer, it recognises
receptor proteins on the surface of cancer cells and binds to them allowing the immune system to identify and destroy them
Treatment of melanoma (a type of skin cancer); the antibody (ipilimumab) binds to a protein produced by T-cells (whose role is to
reduce the immune response) which results in the immune system remain active against the cancer cells
Using monoclonal antibodies as a treatment requires multiple administrations and this can cause problems
Initially the monoclonal antibodies were produced by mice, rabbits or other laboratory animals (as these were easier to produce),
however this triggered an immune response when they were introduced to humans
Scientists have largely overcome this by:
Genetically modifying the antibody polypeptide chains so that the amino acid sequences are now human not mouse or rabbit
sequences
Altering the type and position of the sugar groups (antibodies are glycoproteins) attached to the heavy polypeptide chains to
reflect those found on human antibodies

Types of immunity
Active: Own immune response is activated
Own lymphocytes are activated by antigens
Own antibodies are made
Takes time, not immediate
Memory cells formed → results in long-term immunity
Passive: Immune response is NOT activated
Own lymphocytes cells not activated
NO plasma cells to produce antibodies
 Protection is immediate
NO memory cells formed → only short-term immunity

The 4 types of immunity:

Graph for active natural immunity:

Graph for passive natural immunity (breast-feeding is not considered in the graph below):
Graph for passive artificial immunity:

Effective and ineffective vaccines:

Active immunity

Active immunity is acquired when an antigen enters the body triggering a specific immune response (antibodies are produced)
Active immunity is naturally acquired through exposure to microbes or artificially acquired through vaccinations
The body produces memory cells, along with plasma cells, in both types of active immunity giving the person long-term immunity
In active immunity, during the primary response to a pathogen (natural) or to a vaccination (artificial), the antibody concentration in
the blood takes one to two weeks to increase. If the body is invaded by the same pathogen again or by the pathogen that the person
was vaccinated against then, during the secondary response, the antibody concentration in the blood takes a much shorter period of
time to increase and is higher than after the vaccination or first infection

The primary and secondary response to the same antigen:

Passive immunity

Passive immunity is acquired without an immune response. Antibodies are not produced by the infected person
As the personʼs immune system has not been activated then there are no memory cells that can produce antibodies in a secondary
response. If a person is reinfected they would need another infusion of antibodies
Depending on the disease a person is infected with (eg. tetanus) they may not have time to actively acquire the immunity, that is,
there is no time for active immunity. So passive immunity occurs either artificially or naturally
Artificial passive immunity occurs when people are given an injection / transfusion of the antibodies. In the case of tetanus this is an
antitoxin. The antibodies were collected from people whose immune system had been triggered by a vaccination to produce tetanus
 antibodies
Natural passive immunity occurs when:
Foetuses receive antibodies across the placenta from their mothers
Babies receive the initial breast milk from mothers (the colostrum) which delivers a certain isotype of antibody (IgA)

Comparing Active & Passive Immunity:

How vaccines work

Two modes of vaccination:

1. Mass vaccination
Vaccinate a large number of people at the same time
2. Ring vaccination
Perform contact tracing with infected person
Vaccinate the area of community the person is in / people who was in contact with the person

Aim: Vaccinate a high proportion of the population

→ To achieve herd immunity

Herd immunity:

Mass vaccination results in herd immunity

Less chance of transmission of disease
→ Reduce pool of infected people in the community
→ Fewer people can catch the disease and be source of infection
Protection of those unvaccinated / immunocompromised as disease does not spread
Common barriers to vaccination:

1. Poor response to vaccines

People that are immunocompromised
People who lack protein (malnutrition)
Less antibodies made
2. Pathogens can mutate rapidly
Pathogens can from different strains with different antigens
Memory cells are unable to recognise pathogen that has major changes in antigen structure
3. Some pathogens can escape from immune system (antigenic concealment)
By living inside cells / covering bodies with host proteins / suppressing immune system

How Vaccines Work

A vaccine is a suspension of antigens that are intentionally put into the body to induce artificial active immunity. A specific immune
response where antibodies are released by plasma cells
There are two main types of vaccines:
Live attenuated
Inactivated
Vaccines are administered either by injection or orally (by mouth). When a person is given a vaccine they have been given a
vaccination
The vaccinations given by injection can be into a vein or muscle
Vaccinations produce long-term immunity as they cause memory cells to be created. The immune system remembers the antigen when
reencountered and produces antibodies to it, in what is a faster, stronger secondary response
Vaccines can be:
Highly effective with one vaccination giving a lifetimeʼs protection (although less effective ones will require booster / subsequent
injections)
Generally harmless as they do not cause the disease they protect against because the pathogen is killed by the primary immune
response
Unfortunately there can be problems with vaccines:
People can have a poor response (eg. they are malnourished and cannot produce the antibodies – proteins or their immune system
may be defective)
A live pathogen may be transmitted (e.g. through faeces) to others in the population (ideally enough number of people are
vaccinated at the same time to give herd immunity)
Antigenic variation – the variation (due to major changes) in the antigens of pathogens causes the vaccines to not trigger an
immune response or diseases caused by eukaryotes (eg. malaria) have too many antigens on their cell surface membranes making
 it difficult to produce vaccines that would prompt the immune system quickly enough
Antigenic concealment – this occurs when the pathogen ʻhidesʼ from the immune system by living inside cells or when the
pathogen coats their bodies in host proteins or by parasitising immune cells such as macrophages and T cells (eg. HIV) or by
remaining in parts of the body that are difficult for vaccines to reach (eg. Vibrio cholerae – cholera, remains in the small
intestine)
The principles underpinning vaccinations were discovered by Edward Jenner in the 1700s when he developed the first smallpox vaccine

Live attenuated vaccines

Live attenuated vaccines contain whole pathogens (e.g. bacteria and viruses) that have been ʻweakenedʼ
These weakened pathogens multiply slowly allowing for the body to recognise the antigens and trigger the primary immune response
(plasma cells to produce antibodies)
These vaccines tend to produce a stronger and longer-lasting immune response
They can be unsuitable for people with weak immune systems as the pathogen may divide before sufficient antibodies can be produced
An example of this type of vaccine is the MMR (Measles, Mumps and Rubella)

Inactivated vaccines

Inactivated vaccines contain whole pathogens that have been killed (ʻwhole killedʼ) or small parts (ʻsubunitʼ) of the pathogens (eg.
proteins or sugars or harmless forms of the toxins – toxoids)
As inactivated vaccines do not contain living pathogens they cannot cause disease, even for those with weak immune systems
However these vaccines do not trigger a strong or long-lasting immune response like the live attenuated vaccines. Repeated doses and
/ or booster doses are often required
Some people may have allergic reactions or local reactions (eg. sore arm) to inactivated vaccines as adjuvants (eg. aluminium salts)
may be conjugated (joined) to the subunit of the pathogen to strengthen and lengthen the immune response
An example of a whole killed vaccine is polio vaccine
An example of a toxoid subunit vaccine (where inactivated versions of the toxins produced by pathogens are used) is Diphtheria

Vaccines trigger the primary immune response (T helper cells trigger B plasma cells to secrete specific antibodies) which leads to the
production of memory cells which will give a faster and greater (higher concentration of antibodies) during the secondary response.

Vaccination to control disease

Vaccination to Control Disease

With the exception of the great success story surrounding the eradication of Smallpox following a ten year global initiative in 1980, no
other pathogen has been eradicated globally since
Smallpox was able to be eradicated because a ʻlive attenuatedʼ vaccine was used against the only strain of the virus. There was also
a programme of surveillance, contact tracing and ʻringʼ vaccinations
There are many safe and effective vaccines that do exist against many pathogens and these have managed to push a number of
childhood diseases to the verge of extinction
Vaccines against such diseases as mumps, chicken pox and whooping cough are administered to children as part of an immunisation
schedule and they successfully confer immunity
As a result many childhood diseases are kept at low levels within populations due to herd immunity
Herd immunity arises when a sufficiently large proportion of the population has been vaccinated (and are therefore immune) which
makes it difficult for a pathogen to spread within that population, as those not immunised are protected and unlikely to contract it as
the levels of the disease are so low
Although most vaccinations are given to children there are some vaccines that are provided at later stages in life, for example
vaccinations for tuberculosis (TB) and Hepatitis B are offered to frontline medical workers who have a higher risk of coming into
contact with such diseases in the hospital setting;
Travellers may be advised to take particular vaccines if travelling to areas where certain diseases are endemic such as Yellow
Fever in parts of Africa

How was smallpox eradicated?

An effective vaccine was developed

Same vaccine used everywhere
→ Variola virus – stable, low mutation rate
Used live virus so strong immune response
→ Vaccinia virus was used (a virus similar to Variola virus)
One dose enough to give life-long immunity, no boosters needed
The vaccine is heat stable
Easy to administer
→ Uses bifurcated needle
→ Needle can be sterilised and reused
Mass vaccination was very successful. This is due to:
High percentage of population immunised
→ Low cost needed for mass production of vaccine
→ Many volunteers became vaccinators
→ Result in herd immunity
Infected people were easy to identify
→ Few symptomless carriers
→ Can perform contact tracing and ring vaccination
 → Can isolate cases to prevent

Why isn't measles eradicated already?

MMR vaccine is available

It not eradicated even though there is a high coverage of vaccination

Why?

Some children need several boosters

Measles is highly infectious
→ High percentage cover (93%-95%) needed to achieve herd immunity
→ Must vaccinate whole population and infants <8 months old
→ Not yet done in every country
Difficult for surveillance / to ensure vaccination
→ Due to high birth rates and high migration rates
→ Difficult to give boosters, follow up on cases and trace contacts

Why isn't TB eradicated already?

BCG vaccine is available

It is not eradicated even though there is a high coverage of vaccination

Why?

Vaccination does not work in adults >35yo

High percentage cover (above 90%) needed to achieve herd immunity
→ Not yet done in every country
Difficult for surveillance / to ensure vaccination
→ Due to high brith rates and high migration rates
→ Latent TB is symptomless and found in 1 in 4 people

Why can't we vaccinate against malaria?

No effective vaccines against malaria

Why?

Protoctists are eukaryotes

→ Many more genes than bacteria & viruses
Display different antigens on its cell surface for:
→ Different species / strains
→ Different stages of its life cycle
Parasite changes antigens during infection
→ Different genes coding for antigens switch on during infection
Plasmodium parasite hides in liver and RBCs

Why can't we vaccinate against cholera?

No effective vaccines against cholera

Oral vaccination only gave limited protection as it was excreted

Why?

Many different strains of cholera

→ Bacterium mutates
Vibrio cholerae lives in the host's intestines
→ Beyond reach of antibodies

Types of immunity

1. Artificial active: Vaccination using live, attenuated pathogens

2. Artificial passive: Injection of antibodies against pathogen
3. Natural active: Infection by a pathogen
4. Natural passive: Ingestion of maternal antibodies by an infant through mother’s milk
OR Transfer of maternal antibodies across the placenta to the foetus

Advantages and disadvantages of innate and adaptive immunity

Innate immunity

1. Advantage: rapidly recognises and responds to pathogens

2. Advantage: ability to recognise a large range of antigens through non-specific binding
3. Disadvantage: does not provide a person with long-term immunity against an invading pathogen

Adaptive immunity
 1. Advantage: produces T and B cells that specifically and efficiently target the pathogen and infected cell e.g. antibody-secreting B cells
and cytotoxic T cells
2. Advantage: long-term immunity through development of memory cells
3. Disadvantage: takes longer to respond to an invading pathogen than the innate immune response

Innate immune response

1. Body’s first line of defence against pathogens

2. External mechanisms: physical barriers e.g. the skin and mucus prevent entry of pathogens
3. Internal mechanisms: consist of cellular defences, plasma proteins, inflammatory responses that respond immediately to invasion from
pathogens to contain and get rid of the infection
a. Complement activation: formation of pores (MAC), enhanced phagocytic activity of macrophage
b. Cytokine and chemokine secretion for recruitment of macrophages and neutrophils
c. Inflammation: dilation of blood vessels

Adaptive immune response

1. Macrophage (or specific cells) as antigen-presenting cells (APC)

2. Ref. to T cell recognition through binding with complementary receptors OR Clonal selection
3. Activation of T cells
4. Proliferation / mitosis of activated T cells OR Clonal expansion
5. T (cytotoxic and helper) memory cell formation
6. Description of T cytotoxic cell action e.g. target pathogens and perform direct killing via release of granzyme and perforin
7. Explanation of faster response for second and subsequent contacts e.g. increased number of (specific) T cells increases chance of
coming across APC, more T cytotoxic cells present to destroy (pathogen)
8. T helper cells secrete cytokine to stimulate T cytotoxic cell response / macrophages

Immunological memory

1. Memory T and B cells are produced along with plasma cells

2. There is a greater number of specific immune cells
3. Memory cells are longer lived / remain in circulation
4. Ref. to faster secondary response to give immunity

How mitosis is involved in an immune response

1. Mitosis is involved in clonal expansion of the selected B lymphocytes

2. To form clones / a group of genetically identical cells of selected B lymphocytes
3. Then they differentiate to give rise to plasma cells and memory B cells

Alternative answer

1. Occurs in both primary and secondary immune response

2. Clonal selection and expansion, selected B and T lymphocytes divide by mitosis
3. Mitosis in memory cells for rapid secondary response

Role of T lymphocytes

1. Activated helper T cells release cytokines

2. ;
a. Ref. to activating B cells to proliferate and differentiate into plasma cells and memory B cells
b. Ref. to stimulating macrophages to carry out phagocytosis more vigorously
c. Ref. to activating cytotoxic T cells to destroy infected cells
3. Memory T cells (remain in small quantities in the body) allow for a more rapid secondary immune response

How antigen leads to production of antibodies

1. Macrophages / Dendritic cells engulf foreign antigen by phagocytosis (and carry out intracellular digestion)
2. and present the antigen on their surface
3. Activated helper T cells bind and secrete cytokines
4. B cells are activated and undergo clonal expansion
5. and differentiate into plasma cells that secrete antibodies

How plasma cells produce antibody molecules

1. Genes encoding light and heavy chains are transcribed to mRNA

2. Translation / polypeptides synthesised at the ribosomes / rER
3. Polypeptides enter the rER lumen and undergo protein folding
4. Glycosylation and formation of interchain disulfide bonds of the polypeptides in GA

How antibody molecules are released from plasma cell

1. Secretory vesicles containing the antibody molecules buds off from trans face of GA
2. Then migrate and fuse with the plasma membrane
3. To release antibodies via exocytosis
Role of antibodies

1. Variable region on antibody is complementary and bind to antigens / epitopes on pathogens, resulting in:
2. Neutralisation which blocks the pathogen’s ability to bind to a host cell
3. Opsonisation which promotes phagocytosis by macrophages and neutrophils
4. Activation of complement system and pore formation in the pathogen’s cell membrane

Structure and function of antibodies

Variable region

Variable region forms the antigen-binding sites

Binds / Attaches to antigen
Each antigen-binding site (has a specific 3D shape that) is specific to the shape of one antigen

Hinge region

Disulfide bonds hold polypeptides / heavy chains together

Maintains tertiary / quaternary / 3D shape / structure
Hinge region gives the flexibility for the antibody molecule to bind around the antigen

Constant region

Constant region binds to receptors / cell surface membrane on phagocytes / macrophages

Antigen marking / tagging for phagocytosis / macrophage action
Opsonisation: many phagocytic cells bear receptors for the Fc portion of the antibody and adhere to the antibody-coated bacteria,
leading to engulfing and destruction of the microorganism
Agglutination / Precipitation of antigen: insoluble antigen-antibody complexes are easily phagocytosed and destroyed by phagocytic cells

Difference between humoral immunity and cell mediated immunity:

Humoral immunity produces antigen-specific antibodies and is primarily driven by B cells. Cell-mediated immunity on the other hand does not
depend on antibodies for its adaptive immune functions and is primarily driven by mature T cells, macrophages and the release of cytokines
in response to an antigen.

How humoral immune response enhances action of macrophage

1. Plasma cells produce antibodies / immunoglobulins

2. Agglutination where antibodies clump bacteria to promote phagocytosis by macrophages
3. Opsonisation where antibodies tag pathogen to promote phagocytosis by macrophages
4. Fab region of antibody binds to antigens on pathogen
5. Fc region of antibody binds to Fc receptor on phagocytes to promote phagocytosis

Why infection with one pathogenic strain does not provide immunity to a different pathogenic strain

1. Different strains of bacteria contain different antigens on the surface of the cell
2. Ref. to antigens composed of different sugars
3. Upon infection with one pathogenic strain, the antigens will trigger an immune response whereby receptors of the lymphocytes are
specific / complementary to the antigen
4. Ref. to different antibodies synthesised for different antigens
5. Infection with one pathogenic strain will not provide immunity to another strain because memory cells will not respond to a different
antigen

Why phagocytes act only against bacteria and not human cells

1. Bacterial antigens are non-self / foreign and human cells have self antigens
2. Non-self and self antigens are proteins of different amino acid sequences / Self antigens are encoded by genes in the body
3. Non-self antigens will trigger phagocytosis by APCs (macrophages and dendritic cells)
4. Phagocytes bind to antibodies complexed with non-self antigens / human cells will not have bound antibody

Why some people are not sensitive to a specific pathogen

1. Immunocompromised
2. May not have specific T cells / T cells with specific receptors
3. May have T cells, but low in number and do not come across APC
4. May need several doses to build up sufficient numbers of T cells

Why tuberculosis is known as an infectious disease

1. Caused by a pathogen, bacterium Mycobacterium tuberculosis

2. Transmits easily from infected individual to other uninfected individuals via various modes of transmission - aerosols / airborne
droplets from infected person exhaling / coughing / sneezing / shouting / singing
3. Depending on the environment, these tiny particles can remain suspended in the air for several hours
4. Transmission occurs when a person inhales and the droplet nuclei transverse the mouth or nasal passages, upper respiratory tract, and
bronchi to reach the alveoli of the lungs
5. Person drinks unpasteurised milk / eats meat from infected cattle

How Mycobacterium tuberculosis protects itself from host immune system

1. M. tuberculosis has cell walls that contain mycolic acids

2. Protect bacteria from acids / digestive enzymes / lysosomal enzymes produced by lysosome
3. Inhibit the fusion of the phagosome with lysosomes, hence no phagolysosome is formed and no lysosomal enzymes are available to kill
the bacteria

Why P. syringae can cause disease in host plant by secreting toxins and cell wall degrading enzymes without harming itself

1. Toxins may affect the functioning of membranous organelles / 80S ribosomes not found in the P. syringae
2. Prokaryotic cell wall is composed of peptidoglycan and not cellulose / different compositions of cell wall in plants and in bacteria
3. Specific 3D conformation of the enzymes not complementary to its own bacterial cell wall
4. AVP e.g.
Protective layer / Ref. to capsule
 Cell possesses protective mechanisms that break down toxins so as not to harm itself

Role of antibiotics in treatment of infectious diseases

1. Kill bacteria / bactericidal / cause bacteria to lysel swell and burst OR Bacteriostatic / prevents bacterial growth / prevents bacterial
replication
2. Antibiotics interfere with the modulation of chromosomal supercoiling through topoisomerase (catalyses strand breakage) and rejoining
reactions required for DNA synthesis, mRNA transcription and cell division
3. Prevents protein synthesis (initiation and elongation), inhibit RNA polymerase
4. Antibiotics may also result in protein mistranslation by promoting tRNA mismatch with mRNA codon
5. Antibiotics may also inhibit cell membrane function which results in leakage of important solutes essential for the cell’s survival
6. Prevent spread of bacteria within body / prevent formation of pathogen reservoir for re-infection
7. Do not affect human cells / tissues / not toxic to humans
8. Prevent death / consequences may be fatal if no antibiotic treatment / alleviate symptoms / faster recovery
9. Prevent transmission / spread of disease

Ways to reduce emergence of drug resistance in bacteria

1. Prescribe / Take antibiotics only when (absolutely) necessary

2. Ensure correct / effective antibiotic(s) prescribed / used
3. Complete course / Follow instructions for use of antibiotics
4. Ref. to monitoring situation to check if antibiotic is effective
5. Use other antibacterials / bacteriophages to kill drug resistant bacteria
6. Develop new drugs / antibiotics
7. Ensure / Improve knowledge of healthcare professionals / public
8. Reduce / Control use of antibiotics in agriculture / animals used for food
9. Report patterns of antibiotic resistance
10. Break transmission cycle of drug resistant bacteria e.g. quarantine / isolation

How penicillin acts on bacteria

1. Penicillin weakens the cell wall by binding to and blocking transpeptidases

2. thereby preventing the formation of cross-links between NAM residues in the transpeptidation step during cell wall synthesis
3. Ref. to autolysins weakening the peptidoglycan
4. The weakened cell wall cannot withstand the pressure potential exerted on it by the cell contents and the cell would eventually burst

Different effects of penicillin on Gram-positive and Gram-negative bacteria

1. Penicillin is able to reach the peptidoglycan cell wall of Gram-positive bacteria

2. where it binds and blocks transpeptidases
3. preventing formation of the cross-links between NAM residues in the transpeptidation step in cell wall synthesis in Gram-positive
bacteria
4. While the outer membrane of Gram-negative bacteria stops penicillin passing through to reach the peptidoglycan cell wall, thus penicillin
exerts no effect on Gram-negative bacteria

Why penicillin is less effective on Gram-negative bacteria

1. Presence of the outer membrane prevents / interferes with / protects from entry of penicillin
2. Presence of enzymes in periplasm to degrade penicillin
3. Ref. to presence of proteins in outer membrane that may pump out penicillin
A: presence of efflux pumps
4. Ref. to penicillin being unable to cross hydrophobic region of outer membrane

How HIV causes disease in humans

HIV infection causes destruction of helper T cells by the following mechanisms:

1. Hijacking of cellular machinery and resources towards producing new virus disrupts normal activities needed for cell survival, eventually
causing cell death
2. Budding of a large amount of viruses from the cell surface might also disrupt the cell membrane sufficiently for host cell to die
3. HIV may induce adjacent helper T cells to fuse together forming giant multinucleated cells or syncytia
4. Shortly after syncytia formation occurs, the fused cells lose immune function and die
5. Functional helper T cell levels decline to a critical point

How HIV results in death of host cell

Helper T cells infected with HIV may bind to adjacent uninfected helper T cells / macrophages and fuse together, forming a giant
syncytium
The syncytium may lyse / is recognised and killed by cytotoxic T cells which induce apoptosis of the host cell

OR

Release of HIV particles require the evagination of host cell membrane during budding, resulting in loss of host cell membrane
Induced apoptosis of host cell

OR
 Natural killer / cytotoxic T cells release perforin and granzymes
Inducing apoptosis / osmotic lysis of host cell

Stages where anti-HIV drugs may work

1. At 0 weeks: drugs which bind to viral glycoprotein gp120, thus inhibiting the binding of viral glycoprotein gp120 to host protein CD4 and
viral penetration and entry
2. Between 0-9 weeks: drugs which inhibit the action of reverse transcriptase, thereby inhibiting conversion of viral RNA to DNA and thus
preventing integration
3. At 9 weeks: drugs which inhibit the action of integrase, thereby inhibiting integration of viral DNA into host genome and thus
disallowing persistency of infection
4. After 9 weeks, drugs which inhibit the action of protease, thus preventing the digestion of viral polypeptides into functional proteins
and disallowing packaging of new viruses

How drugs target processes specific to HIV

1. The attachment of HIV is via viral glycoprotein gp120 to CD4 on target cells which confers its specificity. Entry inhibitors blocking this
interaction, prevents HIV attachment and hence entry into the host cell
2. HIV is an RNA virus and it uses reverse transcriptase to synthesize dsDNA using RNA as a template. Reverse transcriptase inhibitors can
stop viral replication
3. HIV DNA is then integrated into the host cell genome with the help of the enzyme integrase. Integrase inhibitors that bind to its active
site or change the conformation of the active site will disable integration
4. HIV DNA is transcribed and translated into viral polyproteins. For the polyprotein to release its functional proteins, it needs to be
cleaved by HIV protease. HIV protease inhibitors will prevent the proper maturation of HIV

Why targeting processes specific to HIV is essential in drug development

1. These are enzymes found in HIV but not in host cells: Reverse transcriptase and protease
2. By disabling these processes, you prevent replication of HIV without harming the human cells
3. Targeting other processes such as transcription / translation will also prevent the host cell gene expression resulting in harmful side
effects

Why it is difficult to treat HIV with a single drug

1. Reverse transcriptase has no proofreading ability resulting in a high rate of mutation

2. Gene mutations in HIV genome will result in changes in primary and hence secondary or tertiary structure (conformation) of viral
enzymes, making the drugs that target them less effective, developing drug resistance
3. High rate of replication results in accumulation of mutations

How simultaneous use of multiple drugs prevents drug resistance

1. Evolution of drug resistance involves genetic changes passing down to the progeny. Chances of simultaneous mutations changing
multiple proteins within the short duration of a single life cycle of HIV is improbable
2. Multiple drugs are able to reduce the viral load better than a single drug. With less viruses circulating, there is proportionally less
mutations occurring and hence lower chance of developing resistance
3. Mutations happen when virus replicates. Anything that can reduce replication reduces mutations
4. Virus is unlikely to be resistant to many drugs (at the same time)

Features of HIV that make it difficult for immune response to eliminate it

1. The HIV double-stranded DNA can integrate into the host genome to form provirus and lie dormant within the host cell, thus it evades
detection by immune response
2. Reverse transcriptase has no proofreading ability hence reverse transcription is prone to errors
3. HIV has very high mutation rate / antigenic drift in the gene coding for viral glycoprotein, hence antibodies can no longer bind
complementarily to the mutated viral glycoprotein
4. Antibodies are not effective against the different strains that arise
5. HIV has very high replication rate which produces more viral particles than can be eliminated (hence antibodies might not be effective
against the high viral load)

Advantages of rapid accumulation of gene mutations in virus

1. Ref. to mutations resulting in production of surface proteins with different 3D conformations

2. Ref. to antibodies being no longer complementary to surface proteins and thus will not recognise and bind to them, rendering vaccines
ineffective OR Ref. to change in antigenicity, and hence able to evade immune response
3. Ref. to wider range of hosts

Outbreak of new viral diseases

1. Existing viruses with high mutation rate as replication of nucleic acid does not involve proofreading
2. Spread of existing viruses from one host species to another
3. Spread of viral disease from small isolated human populations leading to widespread epidemics
4. Genetic recombination between similar strains of viruses

Variation giving rise to new strains of influenza virus

1. Antigenic shift where reassortment of a viral genome with that of a different antigenic type results in the formation of a new influenza
strain
 a. More than one different strain of virus infects a single host cell
b. New virus assembled is a novel combination / reassortment of RNA which arose from the reshuffling of the genome during
packaging of the new virion
2. Antigenic drift where a gradual accumulation of minor mutations results in changes to the haemagglutinin and neuraminidase genes
a. Mutation in the haemagglutinin / neuraminidase gene / viral genome occurs
b. Giving rise to viral proteins with a different specific 3D conformation (such that the viral surface glycoproteins / haemagglutinin
are now more complementary to the specific receptors on the host cells)

Why most people in 1957 were susceptible to new influenza virus

1. Antigenic shift
2. A sudden change in the antigenicity of a virus due to reassortment / reshuffling / new combination of the segmented virus genome with
another genome of a different antigenic type
3. New viral strain has RNA segments 2, 4 and 5 from H2N2 avian virus and segments 9, 11, 14, 15 and 16 from H1N1 human virus
4. New combination of RNA segments causes the virus to change its 3D conformation of HA and/or NA
5. New 3D conformation of the new virus binds more effectively to receptors on the cells of lungs and airways of humans
6. New 3D conformation of the new virus cannot be recognised by antibodies / memory cells / B cells (A: immune system / innate /
adaptive immunity)

Need for annual influenza vaccinations

Ref. to different strains exist, new influenza virus strain can emerge via antigenic drift and antigenic shift

Antigenic drift

Ref. to antigenic drift + spontaneous mutations in the viral genome coding for antigens haemagglutinin and neuraminidase
due to a lack of proofreading of viral RNA-dependent RNA polymerase
causing minor changes in the 3D conformation of haemagglutinin or neuraminidase
Ref. to changes may accumulate to become a new strain

Antigenic shift

Ref. to antigenic shift + two (or more) different strains of the influenza virus infects the same host cell
Reassortment of the viral RNA segments giving rise to a new combinations of RNA segments in new viral particles, hence new
combinations of surface antigens haemagglutinin and neuraminidase arises, a new virus strain results
Annual vaccines contain only 3 strains that are common for that year / season / Ref. annual vaccine does not provide protection for all
strains

How tetanus vaccine prevents clinical symptoms

1. Tetanus vaccine contains inactivated toxin that act as antigens

2. The inactivated toxin is taken up by an antigen-presenting cell (APC) through phagocytosis
3. The APC travels to a secondary lymphoid organ that contains mature naive T and B cells
4. The antigens are processed by the APC and presented on the cell membrane of the APC by MHC class II glycoproteins
5. Antigens on MHC class II are recognised by TCR on helper T cells
6. These T lymphocytes become activated and proliferate
7. Helper T cells interact with and activate B cells
8. which proliferate and differentiate to plasma cells to produce antibodies specific to the toxin
9. Memory B and T cells are formed
10. give long-term immunity against tetanus

Why vaccine not effective with increased age

1. As people age, the thymus shrinks

2. repertoire of naive T cells will be lower
3. Activation of T cells and B cells by the vaccine will be lower
4. Unable to form memory T cells and B cells

Why effective vaccine cannot be produced from heat-treated viruses

1. Viral glycoproteins / recognition proteins denatured due to change in 3D protein conformation through heating, thus cannot trigger
correct immune response

Why it is difficult to design effective vaccine against malaria

1. Parasites evade immune system / mostly stay inside host cells

2. Uses liver cell membrane covering / disguised as ‘self’
3. B lymphocyte response / antibody production not enough / need to stimulate T lymphocytes / cell-mediated response
4. AVP e.g. large degree of variation / change in parasite antigens

Why disease may still result after annual influenza vaccine

Suggest infection by one strain only

1. Exposed to an influenza virus shortly before getting vaccinated OR during the period that it takes the body to gain protection after
getting vaccinated This exposure may result in the person becoming ill with influenza before the vaccine begins to protect the person

Suggest infection by other strains

 2. Ref. to exposed to a flu virus that is not included in the seasonal flu vaccine
3. Ref. to influenza vaccine is made to protect against the 3 strains that research suggests will be most common / likelihood that
prediction for 3 most common strain not being accurate
4. Ref. to existing memory cells / antibodies do not recognise antigens on new strain
5. Ref. to protection provided by vaccination can vary widely, based in part on health and age factors of the person getting vaccinated /
Ref. may not provide protection in people with immune systems that are not fully functioning or developed

Reasons for success in eradicating smallpox

1. Vaccine is thermostable / freeze-dried (longer shelf-life / no wastage)

2. Virus did not mutate
3. Same vaccine could be used everywhere
4. Cheap to produce
5. Ease of production
6. Used a live virus / vaccine gave a strong immune response
7. No need for boosters
8. Ease of administration / Ref. to needles could be sterilised / re-used
9. High percentage cover
10. Ref. to ring vaccination (anyone who could have been exposed to a smallpox patient was tracked down and vaccinated as quickly as
possible)
11. Global effort

Introduction to the Immune System

3 Major Functions of the Immune System

1. Recognises and removes abnormal “self” cells

2. Removes dead or damaged cells and old red blood cells
3. Protects the body from pathogens e.g. microorganisms

Pathologies of the Immune System

1. Incorrect response → autoimmune disease: When mechanisms for distinguishing self from non-self fail and the immune system attacks
the body’s normal cells
2. Overactive response → allergy: When the immune system creates a response that Is out of proportion to the threat posed by the antigen
3. Lack of response → immunodeficiency: When some component of the immune system fails to work properly

Goal of Mounting an Immune Response

1. Immunological recognition: To detect the presence of an infection

2. Immunological effector functions: To contain the infection and if possible eliminate it completely
3. Immune regulation: To keep the immune response under control so that it does not do damage to the body
4. Immunological memory: To protect the individual against recurring diseases due to the same pathogen

Overview of Blood Cells

Contents of centrifuged blood:

3 Key Lines of Defence

Innate Immunity
First Line of Defence: Non-Specific External Barriers

Mucous membranes of gastrointestinal, respiratory and urogenital tracts produce mucus that traps pathogens and other particles
Cilia in respiratory tract sweep mucus out of the body
Acidic pH of skin, stomach and vagina inhibits growth of potential pathogens
Secreted enzymes on skin inhibit microbes attempting to colonise
Commensal microbes colonising the skin and gastrointestinal tract inhibit the establishment of other potentially pathogenic microbes

Second Line of Defence: Innate Immunity

Inflammatory Response

1. Pathogens enter wound

2. Platelets release blood-clotting proteins at the wound site
3. Mast cells release histamine and other mediators, which dilate and increase permeability of blood vessels for entry of immune cells and
complement proteins
4. Macrophages release cytokines, which recruit phagocytes (neutrophils) to the wound site
5. Local accumulation of phagocytes which engulf and destroy microbes and infected cells
*Characteristic features: redness, warmth, swelling, pain

Phagocytosis

1. Neutrophil moves towards the pathogen

2. Neutrophil has surface receptors that recognise and bind to antigens coating the pathogen
3. Neutrophil engulfs the pathogen within a phagocytic vacuole by endocytosis
4. Lysosome fuses with the phagocytic vacuole, forming a phagolysosome
5. Hydrolytic enzymes secreted into the vacuole destroys the pathogen

Fever

Elevated body temperature increases activity of phagocytes and slows bacterial reproduction
Iron deficiency hampers bacterial multiplication
Fever causes cells of the adaptive immune system to multiply more rapidly
 Fever stimulates virus-infected cells to produce interferon, which increases resistance of other cells to viral attack and stimulates
natural killer cells

Adaptive Immunity

Primary and Secondary Immune Response

Upon subsequent exposure to the same antigen, the body mounts a more rapid and effective secondary immune response due to the
presence of memory B and T cells

Adaptive Immune Response

Steps in Adaptive Immune Response

1. THREAT: Antigen evades the first two lines of defence and enters the body
2. DETECTION: Macrophage engulfs and digests antigen by phagocytosis
3. ALERT: Macrophage transports digested pieces to major histocompatibility complex (MHC) markers on its surface, presents the antigen
and binds to specific helper T cell
4. ALARM: Activated helper T cell secretes cytokines, activating macrophages, B cells and cytotoxic T cells
 5. BUILDING SPECIFIC DEFENCES: Naïve B and T cells are activated (clonal selection) and divide rapidly (clonal expansion)
6. DEFENCE: Humoral response – plasma cells secrete antibodies
a. Neutralisation: block antigens from binding to host cell
b. Opsonisation: promote phagocytosis by macrophages and neutrophils
c. Activation of complement system: membrane attack complex forms pores and lyses cell

6. DEFENCE: Cell-mediated response – cytotoxic T cells attack and destroy infected cells
a. Secrete perforin: create pores
b. Secrete granzymes: break down proteins and initiate apoptosis

7. CONTINUED SURVEILLANCE: Memory B and T cells

Summary of Adaptive Immune Response

Active and Passive Immunity

Antigens and Antibodies

Structure of Antibodies:

Viral Infections
Influenza Virus

1. Invasion of first line of defence (entry into host)

Targets: epithelial cells lining respiratory tract
Neuraminidase cleaves sialic acid residues to prevent virus from being trapped in mucus
2. Infection (replication and release of virus)
Initiates antiviral signalling cascades
Released into the airways by budding, which depletes epithelial cell membranes
3. Development of symptoms (dissemination of pathogens)
Excessive mucous production and swelling of epithelial lining
Symptoms: cough, runny nose, fever, chills, sore throat, shortness of breath, muscle or body aches, headaches, fatigue
4. Transmission to other organisms
Airborne (respiratory) transmission

Human Immunodeficiency Virus

Bacterial Infections
Mode of Infection of Bacterial Pathogens

Mycobacterium tuberculosis

1. Droplets containing MTB enter the lung

2. Alveolar macrophages engulf MTB via phagocytosis
3. MTB modifies endosomal compartment to prevent fusion with lysosomes
4. MTB replicates within macrophage, causing macrophage to swell and rupture
5. MTB infects other macrophages, forming tubercles/granulomas – latent stage
Incubation period: a few weeks to several years
6. Tubercle expands and ruptures – active stage
Primary infection in the lungs, secondary infection in lymph nodes, bones and gut
Clinical features: cough, chest pain, shortness of breath, fever, sweating, weight loss

Antibiotics and Bacterial Growth

Typical Growth Cycle of Bacteria

Effects of Antimicrobial Agents on Bacterial Growth

Assaying Antimicrobial Activity

Modes of Action of Antibiotics on Bacteria

Inhibition of cell wall synthesis

1. Penicillin binds and blocks transpeptidases that form peptide cross-links between NAM residues in transpeptidation step in cell wall
synthesis
2. Continued activity of autolysins weakens the cell wall
 3. Cell wall cannot withstand pressure and cell bursts – bacteriolytic

Inhibition of nucleic acid structure and function

Sulfonamides and trimethoprim inhibit folic acid metabolism → block synthesis of nucleotides
Quinolones inhibit DNA gyrase → block DNA replication
Rifampin inhibit RNA polymerase → block transcription

Inhibition of protein synthesis

Aminoglycosides cause misreading of RNA

Tetracyclins block attachment of tRNA on A site
Chloramphenicol prevents formation of peptide bonds
Erythromycin inhibits translocation

Interference with cell membrane structure or function

Polymyxins affect integrity of the membrane, causing it to become leaky

Vaccination
Concept of Vaccination and Herd Immunity

Vaccination stimulates immunity without causing disease by exposing the body to antigens similar to those on a pathogen; this
simulates the real infection and induces a host immune response
Types of vaccines: Live attenuated, inactivated, toxoid, subunit, recombinant, DNA vaccines
Herd immunity: Vaccination of a high enough proportion of the population can break the disease transmission cycle and provide a
measure of protection for individuals who have not developed immunity
Reduces natural reservoir of infected
Reduces transmission
Protects non-immune or those who respond poorly to vaccines

Eradication of Smallpox by Vaccination

Key Reasons for Success in Eradicating Smallpox

Virus did not mutate hence the same vaccine could be used
Virus only infects humans which made it easier to break the transmission cycle
Low cost and ease of production of vaccine
Use of ‘live’ vaccine which induced a strong immune response
Vaccine was freeze-dried with a long shelf-life
Infected people were easy to identify due to visible rash
Ring vaccination: anyone potentially exposed was tracked down and vaccinated as quickly as possible
Global effort and high percentage cover

Benefits and Risks of Vaccination

Leukemias
Leukaemias

Leukaemias are cancers of stem cells which differentiate into blood cells.
Originate in bone marrow
Cause large increase in immature, non-functional blood cells.
More lymphocytes than normal
No large nuclei in lymphocytes
People with leukaemia have severe lack of normal blood cells.

Leukaemias are cancers of stem cells that produce blood cells. These cells do not differentiate and cause a large increase in non-functional
blood cells.

Leukaemia originates in the bone marrow, where stem cells are produced.

People with any leukaemia have a severe lack of normal blood cells.

There are four main types:

1. Myeloid Leukaemia
Cancer of stem cells that go on to form erythrocytes, platelets, neutrophils.
2. Lymphoblastic Leukaemia
Cancer in the cells that divide to form lymphocytes
3. Acute Leukaemia
These develop very quickly and require immediate medical attention
4. Chronic Leukaemia
Develop over a long period of time; monitored to give the most effective treatment

Autoimmune disease
Autoimmune Disease - Failure of immune system to distinguish self from non-self, so antibodies are produced against self antigens.

An autoimmune disease is the failure of your immune system to distinguish self from non-self, so antibodies are produced against self
antigens.

Myasthenia Gravis

An example of autoimmune disease is myasthenia gravis (MG).

In MG, specific B-lymphocytes that produce antibodies against self antigens are not destroyed during development.
Antibodies are produced against self antigens.
These B-lymphocytes produce antibodies against the cell surface receptors on muscle cells, which bind to and block these receptors.
Neurotransmitter molecules, such as acetylcholine, can no longer bind to these receptors. Thus, impulses cannot be sent, causing
muscle weakness & fatigue.
Myasthenia Gravis (MG)

Example of autoimmune disease.

Specific B-lymphocytes produce antibodies against cell surface receptors on muscle cells
Not destroyed during development.
Antibodies bind and block acetylcholine receptors
Neurotransmitters no longer bind.
No impulses sent, causes muscle weakness & fatigue