Lecture 1

Lymphocytes start on jawless fish (lamprey)

Fruit Fly has toll receptor
Humans have more complex TLRs, we have pattern recognition receptors that are more
complex version of toll like receptors.

CDR-> Complementary determining region -> region of lymphocyte receptor for antigen
(participates in antigen binding)

GALT -> gut associated lymphoid tissue -> choose if it is foreign pathogen or food

70 percent of immune cells is going to the gut

Thymus and spleen developed in more advanced organism, critical to mammalian immunity

Peyers patches -> secondary lymphoid tissue in the small intestine

Complexity increases as evolution goes along

(key thymus, spleen, dendritic cells)

Thymus – t lymphocytes go to mature to lymphatic system

Spleen (massive bag of b and t lymphocytes and big filter of toxins)– volume of blood goes
to, checked, removed dead red blood cells, and contains plup (produce antibodies)

DC -> dendritic cells -> ingest infectious agents in peripheral tissues, which then goes to the
lymphoid tissues and present particles to present for T lymphocytes
From Innate to adaptive
Act as Phagocytes in lymphoid tissue -> becomes APC

We have capacitiy to hold much more immune memory compared to sharks and primitive
fish

Number of cells in immune system is more than in brain (10^12 cells, more than 10 cell
types, and infinite number of connections)
Everything Start from hematopoietic stem cell -> it is pluripotent (in bone marrow)
Pluripotent – able to produce all different cell types
Chemical signals are sent out to produce the needed cell type to deal with the infection
Cytokines -> chemical signaling produced by cells to go in blood streams to recruit cells
produced by a hematopoietic stem cell

Where is the immune system? In blood

Blood
Plasma = 95% water with dissolved proteins, glucose,
clotting factors, electrolytes, hormones, carbon dioxide
+ oxygen
Serum = plasma without clotting factors

- Lymphatic system facilitates innate and adaptive connections

- White blood cells circulate with blood
- Some WBCs and quite a bit of plasma leave the capillaries in tissues
- Circulation of plasma and WBCs through interstitial space/tissues and back via
lymphatics to lymph nodes allows surveillance of tissues for foreign molecules and
pathogens
- Around 70% of the immune system is associated with the gut
- critical interactions inside lymphatic system, not the blood (spleen and thymus,
tonsils etc.)
Cells of the immune system are mobile

A Neutrophil
B Eosinophil
C Basophil
D Neutrophil
E Basophil
F Plasma cells
G H I B and T-lymphocytes
J K L Monocytes/Macrophages

- Immune cells in blood are sometimes called Granulocytes

- Neutrophils, eosinophils, basophils -> phagocytic cells that are recruited. innate
immunity. Basophil and eosinphils produce histamines that degranulate. They attack
variety of pathogens by phagocytosis or with anti-microbials and histamine released
after degranulkation

- Plasma cells, derived from B lymphocytes, secrete large amounts of antibodies

- B and T lymphocytes, orchestrate the adaptive immune response and provide

immunological memory

- Monocytes/Macrophages, phagocytose and regulate many aspects of innate and

adaptive immunity

- Dendritic cells switch innate to adaptive immune system

What does the immune system do?

Pathogens and pathogen recognition

- Including Bacteria, fungi, parasites, viruses, cancer cells

- Pathogens are usually covered in a diversity of molecules
- Each has its own molecular signatures (antigens) known as Pathogen-Associated
Molecular Patterns (PAMPs). Usually isn’t in cells from our own body.
- Immune cells have invariant receptors on their surfaces known as Pattern
Recognition Receptors that recognize many of these PAMPs
- Once a pathogen is recognized, a cascade of events occurs activating an Immune
Response to destroy it

Innate and Adaptive Immunity

- Innate -> front line -> looking for foreign molecules -> good at containing low level
infection
- Adaptive -> slower -> memory from previous infection (takes a while)
- Dendritic cell -> take info from innate to adaptive -> move to lymphatic system to
activate adaptive immunity

Lungs covered in alveolar macrophages

- Binding of PAMPs by PRRs on phagocytic cells (e.g. macrophages in peripheral

tissues) leads to an INNATE immune response. This innate response is FAST and
doesn’t require immunological memory
- The ADAPTIVE immune response is slower and is activated by a cascade of chemical
signals from sites of infection, and physical interactions with phagocytic cells of the
innate immune system known as DENDRITIC CELLS that migrate from sites of infection
into the lymphatic system to activate LYMPHOCYTES

The extent to which the two arms of the immune system are activated depends on the
severity, context and duration of disease - innate immunity is usually all that’s needed for a
minor cut or splinter for example
Cytokines and Chemokines

- Macrophages engulfed bacteria -> send cytokines and chemokines to recruit

neutrophils and activate adaptive immunity
- Cut allow cells to leak out, so neutrophils leak through the junction
- Other inflammatory mediators go through also
- IL-1, goes to hypothalamus, goes higher in temperature body, as bacteria don’t like
heat
- Signaling molecules known as Cytokines and Chemokines released at sites of
infection by resident macrophages recruit neutrophils from the bloodstream leading
to localised inflammation

Lymphocytes

- Adaptive immune system

- A lot of diversity, mutates to
produce a newer antibody
- NK cells -> good for cancer
cells and virus infected cells
(regard as innate as they
don’t require previous
activation)
Lymphocytes and Adaptive Immunity

- Lymphocytes are key players in adaptive immunity

- They express a vast repertoire of recognition receptors: B-lymphocytes express
antibodies as B Cell Receptors (BCRs) while T-lymphocytes express T Cell Receptors
(TCRs)
- During an adaptive immune response, B- and T-lymphocytes with receptors specific
for particular pathogen molecules proliferate
- Clonal selection and expansion forms the basis of protective immunity and
immunological memory by which the body is protected against future attack
- As B cells mature, there is a high affinity of antibodies

Molecular and cellular interactions, antigen recognition, cell-signalling, cytokines and

chemokines

- In some people it leads to the overproduction of cytokines -> influenza

- But in most, it doesn’t because the immune system has evolved to be a complex but
very robust system that maintains homeostasis while at the same being able to
rapidly commit to particular courses of effector action to counter disease threats
- All of this is dependent on exquisitely-specific molecular interactions between:

Antigen receptors and antigens

Cytokines/chemokines and their receptors
Cell-cell interactions
Lecture 2

Foreign activators
– Have an understanding of the types of molecule that interact with the
immune system
– Have an understanding of how the immune system recognises these
molecules
– Have detailed understanding of some important recognition events

The immune system recognises foreign activators

What do we mean by foreign? foreign means “non-self”
• Molecules that originate outside of the body
– microbes
– allergens
– foreign tissues
– Transplants
– Cancer Cells
– Outside of body at birth

The immune system recognises non-self

• But usually not small molecules like most chemicals
• Molecules with a size of less than several thousand daltons are poorly recognised
• Polymers like proteins & polysaccharides are recognised well

When the system fails

- Non-responsiveness (tolerance) - immune system fails to recognise foreign
activators (Aids)
- Over-responsiveness - Immune system causes disease (autoimmune disease, sepsis)
- Tumours not seen by antigens

The Two Arms of the Immune System

- The innate and adaptive
arms of the immune system
- Innate -> front line
- Adaptive -> more targeted
(needs innate to feed info
to start)
- use receptors to recognise
foreign (non-self) molecules
- use receptor to
communicate within the
immune system
- Receptors are critical and
are either released by
immune cells or on immune cells
- Also involved in transferring knowledge from one cell to a different cell
Receptors that recognise non-self

Innate system

Soluble receptors
Secreted from cells and found in blood, plasma, lymph, mucosal secretions etc

Cell surface receptors

Found on macrophages, neutrophils, lymphocytes and on other somatic cells

- Can discriminate self and non-self

- Receptors encoded in germline. Limited diversity
- Typically recognise structures common to different microbes

Innate – limited diversity, precoded to recognize foreign activators. Recognise structures

common to different microbes

Adaptive system
Soluble receptors
Secreted from cells and found in blood, plasma, lymph, mucosal secretions etc

Cell surface receptors

Found on macrophages, neutrophils, lymphocytes and on other somatic cells

- Can discriminate self and non-self

- Receptors encoded by gene recombination – massive diversity
- Typically recognise structures unique to different microbes

Adaptive – take its time to evolve, more powerful, more potent immune cells, receptor are
generated by recombination of genes, massive diversity. Recognise structures unique to
different microbes

Receptors are also involved in signalling between cells of the immune system

- Cytokines; chemical signals that regulate the immune system

- Antigen presenting cells, bind to t cells and produce cytokines that turn on immune
cells
When receptor signaling is out of control……

- Autoimmune disease
- Sepsis is a life-threatening condition that arises when the body's response to
infection causes injury to its own tissues and organs.
- Common signs and symptoms include fever, increased heart rate, increased
breathing rate, and confusion.
- Immune system responds too powerfully to the infection
- Overproduction of cytokines and stimulating pathways
- Hard to treat
- Immune system over responds to a foreign activator

1918 Spanish Flu

- Most devastating influenza outbreak ever recorded

- Killed 20-40 million people
- Reduced average lifespan in the US by 10 years
- 20-30s mainly affected
- These people have the best immune systems, which over responded to the antigens
- Overproduction of cytokines

TGN1412 - Theralizumab

- An antibody to CD28 on T-cells

- Experimental drug intended to cause a mild activation of the immune system
- Shown to be effective in animals
- Drug design to target CD28 on t cells
- Aimed to gently stimulate t cells
- Respond to infections

- Used at 500 times lower dose than tested in animals

- caused a cytokine storm
- 6 volunteers hospitalised, 4 suffered from multiple organ failure
- Some volunteers lost fingers and feet
- Activation of cd28 leads to different set of responses to animals
- Over activation of cytokines
- Caused Disseminated intravascular coagulation - is a condition in which blood clots
form throughout the body, blocking small blood vessels
Recognition of non-self - a) innate system

The innate system recognises molecules that are common to microbial pathogens but
absent from the host
- Pathogen-Associated Molecular Patterns (PAMPs)
- The innate system is pre-coded to recognise a limited repertoire of molecules
- This enables it to respond quickly, but only to provide a low-level response

Examples of molecules recognised by the innate system

These molecules usually have multiple of these PAMPs

Cell wall components

- Peptidoglycan (bacteria)
- Lipopolysaccharide (gram positive bacteria)
- Phospholipomannans (fungi)

Nucleic acid
- CpG motif DNA (bacteria)
- ssRNA (viruses)
- Ds RNA (viruses)

Conserved surface proteins

- Flagellin (bacteria)

Conserved stress proteins

- Heat shock proteins (bacteria)

Pattern Recognition Receptors (PRRs)

Soluble receptors
- Natural antibodies
- Complement
- Pentraxins
- Collectins
- Ficolins

Cell receptors
- Toll-like receptors
- NOD-like receptors
- RIG-like receptors
- C-type lectin-like receptors
- Scavenger receptors
- N-formyl met-leu-phe receptors
 Toll-like receptors

- Cell surface receptors

- Horseshoe receptor in extracellular domain for
ligand recognition
- Intracellular domain for signaling
- Dimersation is needed for singalling into the cells
- 2 toll like receptors back to back

There are different toll like receptors that can recognize different molecules. They have
different dimers.
TLR signalling and inflammatory
responses

- Over activation of toll like

receptor, and too much of these
become dimerized, then there
might be sepsis
- A part of PRRs
- TLR4 recognizes LPS in gram
negative
- Too much activation - cytokine
storm

Recognition of bacterial molecule by TLR

-> dimerization of the TLR -> signalling
and inflammatory responses

LPS signalling

TLR4 recognizes LPS in gram neg

LPS->LPS binding protein -> CD14->loads CD14 to the ectodomain to TLR4, binds it to MD2

Homodimerisation occurs when TLR4 MD2 and CD14 bind together

Toll IL-1 Receptor (TIR) activated after homodimerisation
MYD88 recognises the change of TIR, then binds to TIR
Then activates NFKb pathway by phosphorylating iNFKb (inhibitor)
Major inflammation by producing inflammatory cytokines

How is self-recognition avoided ?

– The innate system recognises molecules which are never found in mammals

– CpG motif DNA (bacteria)

– lipopolysaccharide (gram negative bacteria)
– phospholipomannans
– peptidoglycan (bacteria)
– phospholipomannans (fungi)
Recognition of non-self b) adaptive system

The adaptive system recognises antigens (motifs) that are unique to an individual
pathogen

- It takes time to develop and mature, but can provides a very powerful protective
response
- Exploited in vaccines
- Power to discriminate to a specific one

Most common types of antigen recognised by the adaptive system

• Proteins
• Polysaccharides

Soluble receptors
- Antibodies

Cell Receptors
- B cell receptors
- T cell receptors

Much smaller panel of different receptors, Antibodies released (big diversity)

Antibodies recognise discrete epitopes on a protein antigen

- Dissolve and change the tip so they can

recognize slightly different molecules
- Complementary determining regions (CDR)
is the bits that can evolve and change and
recognise different molecules
- Differences are in the CDR
- CDR physically interacting with the antigen
- Complementarity-determining regions
(CDRs) are part of the variable chains in immunoglobulins (antibodies) and T cell
 receptors, generated by B-cells and T-cells respectively, where these molecules bind
to their specific antigen.

Epitopes recognised on C. perfringens epsilon toxin

- Enterotoxaemia in lambs is caused by C. perfringens epsilon toxin

- Common in Domesticated live stock
- Want to find out which part of the regions of this disease are recognised by
antibodies
- a whole range of antibody which can recognize a different part of a single protein

Where does the myriad of antibodies each specific for a different epitope come from?

- B-cell possesses gene for one antibody type that recognises one epitope
- The different genes are generated by random recombination in germ cells
- different antibodies recognise different epitopes
- Each b cell(antibody producing cells) is a clonal population from a 1 particular
recombined gene that produce 1 specific antibody
- A trillion types of b cell capable of producing a slightly different antibody for a type
of antigen
- Generates receptors from recombination events
-
Progenitor cells (stem cells), step down from pluripotent stem cells. Hematopoietic stem
cells.

The generation of B-cells with encoding different antibodies occurs in the bone marrow

- B cells are made in the bone marrow (recombination events, cloning)

How many different epitopes can be recognised ? (how many different types of B-cell are
there)

How is self-recognition avoided?

- Clonal deletion eliminates self-recognition by the adaptive response
- Clonal deletion deletes any potential b cell where it can recognize self-antigens
(deleted in the bone marrow, like a filter)
- (where there is a recombination event that recognizes self)
- Deleted in the bone marrow, like a filter that would remove antibodies that react to
own tissues
Clonal deletion eliminates self-recognition
- B-cells mature in the bone marrow
- self antigens are also present in the bone
marrow
- B-cells which produce antibodies against
these antigens (epitopes) are eliminated

Lecture 3

Innate immunity

Innate system

Soluble receptors
- Antibodies

Cell Receptors
- B cell receptors
- T cell receptors

Soluble receptors
- Natural antibodies
- Complement
- Pentraxins
- Collectins
- Ficolins
Cell receptors
- Toll-like receptors
- NOD-like receptors
- RIG-like receptors
- C-type lectin-like receptors
- Scavenger receptors
- N-formyl met-leu-phe receptors

Pathogens

Vertebrate bodies are under constant attack by micro-organisms that normally co-exist
harmlessly with their hosts:
• Viruses
• Fungi and Parasites
• Bacteria

Capable of colonising the cytoplasm, intracellular vesicles of cells and interstitial spaces, in
the blood or lymph, or on epithelial surfaces

Breaching the Body’s Defenses

 The first critical barrier to infectious disease is the skin

 Cuts, abrasions, more serious wounds and burns expose body tissues to bacterial
and fungal infections that can be fatal
 The main portals of entry are the mucosal epithelia of the gastrointestinal,
respiratory and urogenital tracts

Immunity
 Immunity requires the recognition and elimination/containment of infectious
organisms
 Most of the mechanisms of front-line defence against infectious agents are provided
by the innate immune system:
 Consists of molecules and cells that distinguish host cells from those of infectious
agents by recognizing conserved constituents of microorganisms (Pathogen
Associated Molecular Patterns - PAMPs).
 Activated within hours of contact and efficacy not significantly increased by previous
exposure
Immune Cell Differentiation

 Cells of the innate and adaptive immune

systems are derived from the pluripotent
hematopoietic stem cell. The HSC divides
to replace itself and produce a progenitor
cell with the potential to give rise to all of
the hematopoietic lineages, by
generating further progenitor cells
committed to progressively narrower
ranges of differentiated fates
 Stimulus received from site of infection ->
Cytokine release to tell hematopoietic
stem cell to go down lineage ->
Depending on strength, will determine
where it will end up
 Gets signal -> produces clones of itself ->
go down pathway -> migrate to site of
infection

Immune Cell Differentiation

 Both red and white blood cells are thought to be derived from a common
hematopoietic stem cell (HSC) via committed progenitors that give rise to the
erythroid, myeloid and lymphoid lineages
 [The erythroid lineage gives rise to the erythrocytes and to megakaryocytes which
shed fragments that form the platelets that initiate blood clotting] RBC, erthrocytes,
megakaryocytes, form platelets, blood clotting
 The myeloid lineage gives rise to the phagocytic and inflammatory cells of innate
immunity. phagocytic and inflammatory cells of innate (innate) – neutrophils
macrophage dendritic cells
 The lymphoid lineage gives rise to the T and B cells of adaptive immunity, and to
natural killer (NK) cells which are specialised cytotoxic cells (innate)

Lymphoid or myeloid route

 Myeloid - differentiate into neutrophils, eosinophils, basophils, monocytes, mast
cells. monocytes differentiate into macrophages or dendritic cells
 Can have myeloid or lymphoid dendritic cells (just remember myeloid)
  Innate immunity cells -> neutrophils, eosinphils, basophils, macrophages, dendritic
cells, mast cells
 After differentiation, migrate to tissues, go to hunt at site of infection
 Macrophages, once differentiated, will sit in the tissues, sentinal cells
 Mast cells, also sentinel cells, involved in allergic reactions, lie in muscus membrane
waiting for allergens. Dendritic cells relay cell to send info between adaptive and
innate system. Used to turn on adaptive

Hematopoietic Stem Cell Lineages

Myeloid lineage - Basophils, Mast cells and Eosinophils

 Mast cell and basophils produce histamines

 Eosinophils and those 2 are important for allergies and parasites. Work by
degranulation of eosinophil peroxidase, ribonuclease (RNase), deoxyribonucleases
(DNase), lipase, plasminogen, and major basic protein.
 Basophils, mast cells and eosinophils play a special role in the protection of epithelial
surfaces, especially the mucosa of the gastrointestinal, respiratory and urogenital
tracts
 Mast cells have a sentinel role; basophils and eosinophils are circulating cells
recruited from the bloodstream
 Contain granules that contain toxic components. They receive signal that
degranulate. Spew content to the outside. Release Histamines and other proteases –
coughing, sneezing, vomiting. First cells of the immune systems. Important in allergic
reactions, parasites. Fungal spores, seasonal allergies to dampen down the reaction
 Basophils and mast cells also release histamines that mediate allergic and
inflammatory responses such as coughing, sneezing and vomiting
 These expulsive responses may be mechanisms that evolved to expel e.g. parasites

Myeloid lineage - Neutrophils, Macrophages and Dendritic cells

 Phagocytes
  Ilya Mechnikov discovered Phagocytosis (introduced small throsn from tangerine
tree to larvae of starfishes. Thorns surrounded by mobile cells)
 The PHAGOCYTIC cells of the immune system comprise of neutrophils and
macrophages which are effector cells specialised to internalise and destroy micro-
organisms, and dendritic cells, which internalise micro-organisms for presentation to
T lymphocytes of the adaptive immune system
 These are phagocytic cells that deal with infection, migrate and engulf foreign
particles and deal with them

 Neutrophils and macrophages are effector cells

 First cells that will deal with infection is the sentinel macrophages
 Neutrophils will be produced through signal sent by macrophages if macrophages
cannot destroy foregin particles (neutrophils stronger)
 Neutrophils (granulocytes) are the front-line effector cells of innate immunity
 After differentiation, they circulate for a few hours before entering tissues to engulf
micro-organisms and destroy them via microbicidal products stored in vesicles
 Neutrophils destroy internalised micro-organisms by delivering them to the cytotoxic
compartments inside the cell, these cells typically function in immune defence
against pathogens that are too large to be internalized. On activation, they release
the contents of their granules to the exterior, thereby either creating an
environment hostile to invading organisms or directly killing the pathogens

 Macrophages are long-lived cells that provide ‘immune surveillance’. They are
derived from monocytes that circulate in the blood, differentiating as they leave the
bloodstream
 Like neutrophils, they ingest and destroy micro-organisms

 Dendritic cells -> like macrophages, but not completely destroy cell, but break them
apart to their constituent part, move the compoennent to the outer and display
them on the outer, move in adaptive immune system to look at the broken down
particles to decide if they turn on adaptive (ANTIGEN PRESENTING CELLS). Critical
cells between innate and adptive immunity
 Dendritic cells are involved in the induction of adaptive immunity. As immature cells
they operate as phagocytes but rather than destroying the micro-organisms they
ingest, their function is to display the ingested particles on their surface for
recognition by T lymphocytes
 NK cells are also Innate

 Granulocytes – neutrophil eosinpohil and basophil and mast cells. Granulocytes are a
category of white blood cells characterized by the presence of granules in their
cytoplasm.
 Leukocytes – All immune cells -> WBC
Macrophage Receptors
  Macrophages display an extensive array of receptors that recognise conserved
components of micro-organisms as well as scavenger receptors that that recognize
particles released by dead or damaged tissues.
 They also have receptors for complement and for antibodies.
 A similar array of receptors is expressed on the surface of dendritic cells.
 They have PRRs
 Know 3 of them
 FC receptors to bind to antibody for antibody dependent binding, uptake and killing
 C type Leptin receptors for pathogen recognition, ICAM adhesion, Mostly on DC
 TLR – responses to PAMPs, mostly Peptidoglycan (TLR2), LPS (TL4 and MD2), ssDNA,
dsDNA

Activation of the Innate Immune System

 Innate immunity is invoked either directly through recognition of micro-organisms by

the innate immune system itself, or indirectly through recognition by cells of the
adaptive immune system that then activate innate immune defences
 Macrophages have Massive array of receptors
 Receptor recognizes foreign receptors, receptor for specific PAMP, activaes
macrophage, migrates toward pathogen, take up the whole bacterium into the cell
including the receptor for destruction

Indirect Activation of the Innate Immune System

 A lymphocyte of the adaptive immune system produces antibodies whose variable

regions which recognise a surface component of the bacterium.
 A non-variable region of the antibody is then recognised by a receptor of the
phagocyte, which in turn is activated to engulf it. In this way, bacteria that have
masked the conserved components can be recognised and destroyed by the innate
immune system.
 B cells will produce antibodies, pepper the outside of the bacteria and picked up by
the FC receptor in the macrophages.
 Indirect mechanism, use antibody to cover cell or opsonize it, phagocyte dock with
antibody and move it into the cell.
Phagocytes and Phagosomes

 Opsonin - a general term for soluble (humoral)

components of the immune system e.g. IgG1 that
coat micro-organisms and stimulate uptake by
phagocytes. Covering something with antibodies
(opsonin)
 Recognise foreign particle, opsonized, Fc receptor on
the outside macrophage or neutropjil recognizes it.
 Goes through actin polymerisation and drags the
whole thing into the cell in to the phagosome
 And then moves it in to the lysosome, (called
phagolysosome)
 Has toxic components such as ROS to destroy it

Phagocytes and Cytokines

The phagocytes of innate immunity play a pivotal role in immune

responses in two ways:
1. On activation by microbial surfaces they release cytokines and
chemokines (chemotactic cytokines) that amplify the response to infection.
Cytokines and chemokines are signalling molecules. They increase the permeability
of blood vessels and recruit additional cells and molecules of the immune system to
sites of infection - the inflammatory response
2. They activate the adaptive immune response

Chemokine -> Lay a trail where cells will migrate to the site of infection
Cytokines

Know a few
 IL-1
 IL-3
 IL-4
 IL-10
 IFNy
 TGFb
 TNFa

The Inflammatory response

- tissue of body kept under surveillance by memory t cells, if they don’t meet any
antigens they return to the circulation via lymphatic system
- If there are antigens, taken up by APC, presenting at cell surface as MHC peptide
complex, memory t cell encounter these become activated, release cytokines, mainly
TNFa that act on local blood vessels, activate the vascular endothelium, tight
junction loosen, allow cytokines to pass out of blood vessels to cause local swelling,
leakage causes local tissue to become redden, activated endothelium also expresses
adhesion molecules that allow phagocytes to migrate out of blood vessel, takes 1-3
days
- Called delayed hypersensitivity response
- These Cells phagocytose antigens, macrophages can act as additional APC
- Memory and effector t cells also recognize the adhesion molecules expressed by the
vascular endothelium and migrate into the tissue, to continue the process of
inflammation
Cytokines and Chemokines from Macrophages

- Activated macrophage that engulfed the pathogen which will activate these
cytokines
- Don’t need to learn any other cytokines besides these

Cytokine Signalling

- Cytokine receptors have 2 chains

- each have an extracellular cytokine binding domain and an intracytoplasmic domain
which binds to a member of the family of protein tyrosine kinase called JAK (Janus
Kinase)
- In the absense of the cytokine, the 2 chains do not stay binded
- Cytokine binding to the receptor to stablize the heterodimer and brings together the
JAK that are bound to the cytoplasmic portion of each chain
- The JAK kinase are then able to phosphorylate the cytoplasmic tail of the cytokine
receptors
- STAT molecules (Signal transduction and transcription or) bind to the
phosphorylated receptor chains and are then phosphorylated by the JAK
- The addition of a phosphate to the STAT allows them to dimerize and migrate into
the nucleus where they can activate gene transcription
Cytokines and Neutrophil Chemotaxis

Chemotaxis - movement of a motile cell or organism, or part of one, in a direction

corresponding to a gradient of increasing or decreasing concentration of a particular
substance.
Chemotaxis is the movement of an organism in response to a chemical stimulus
Aid in migration of phagocytic procress
IL8 – chemokine

Allow neutrophils to get to site of infection, identify there is an infection underneneath, and
squeeze through vascular system to get to the cells to help the macrophages – rolling
adhesion

Rolling Adhesion

- Blood vessel endothelial at site of infection express selectin molecules, P selectin and
E selectin
- leukocytes such as neutrophils express the liagnd for selectin in the form s-Lex
- binding of the endothelial selectin to s-lex is weak and cannot hold the cell against
the flow of blood
- Instead the cell rolls along the wall of the blood vessel making and breaking many
reactions with the selectins
- Not randomly shoved along blood vessel, Recognising tethers along the inside of the
blood vessel that allows it to slow down as the signal gets stronger and stronger
Neutrophil Rolling
- Leucocytes only leak through veins and not arteries
- Artery – blood right to left
- Vein – blood flow left to right
- Leucocytes are WBC that help fight infection
- at sites of injury or infection or inflmmiton, cytokines are released and stimualte
endothelial cells that line adjacent blood vessels
- The endothelial cells express surface proteins called selectins
- selectins bind to carbohydrates displayed on the membrane of the leukocytes
causing them to stick to the wall of the blood vessels
- this binding interaction is of low affinity that the leukocyte can roll along the vessel
wall to in search for point to exit the vessel
- there they adhere tightly and squeeze in between endothelial cells without
disruption the endothelial walls then crawl out of the blood vessels into the adjacent
connective tissue
- Leukocytes only adhere so surface of veins and not arteries

Recruitment of Neutrophils to Inflammatory Sites

- Not just relying on cytokines and chemokines
- Other components released by bacteria that are really potent activators of
neutrophils
- Fluid matrices contain antibacterial peptides and toxic proteins that are
constitutively secreted and further induced in response to cell wall components such
as lipopolysaccharide
- tears -> antibacterial peptides and toxins, always secreted always there, but when a
bacterium appears with LPS, they are massively upregulated

- b-defensins in lung and skin

- a-defensins in Paneth cells of the intestinal tract
- Lysozyme in fluids bathing the cornea (tear duct) (make bacteria exploid)
- Cathelicidins (another group of antimicrobial defensins) are produced by activated
neutrophils and epithelial cells - in addition to antimicrobial activity they trigger
signaling through the formyl peptide receptor, an innate immune recognition
element, thereby aiding recruitment of neutrophils to inflammatory sites

Formyl-Methionyl Peptides and Phagocytosis

- fMLP potent activator of that receptor on the outside of neutrophils

- Bacterial peptides e.g. formyl-methionyl-leucyl-phenylalanine (fMLP) are powerful
chemoattractants and activators of neutrophils
- Derived from NH2-terminal extensions of newly synthesized polypetides
- fMLP directs the transport of secreted proteins that possess NH2-terminal signal
peptides
 - Cleaved by a signal peptidase following polypetide transport and released into the
extracellular space
Lecture 4 - adaptive immune system

Immune Memory

- The adaptive immune system consists of T and B lymphocytes

- The T lymphocytes and immunoglobulins (antibodies) that B-lymphocytes produce
can recognise an unlimited number of different targets
- Become effective only after a delay of 2 to 4 days on first encounter with a
microorganism
- Lymphocytes and lymphocyte products specific for a given organism then persist as
immune memory and are rapidly protective on re-exposure to the same infectious
agent
- Can recognize pretty much antigen
- Delay – takes few days for adaptive immunity to kick in
- Innate cells send signals to adaptive immune system in lympatic tissue to start
- T and B cells persist as memory sitting in the lymphatic system
- Vaccines

What does vaccination do?

- The ability of the body to remember an infectious agent to

- which it has been exposed is the basis for natural and artificial
- immunity against a disease
- Vaccination initiates a primary immune response, generating
- memory cells without making the person ill
- Later, if the same or very similar pathogens invade the body,
- specific memory cells already exist
- They recognise the agent and produce massive number of
- lymphocytes and immunoglobulins that overwhelm the invaders
- Inactivated pathogen/antigen, introduce it to the body, activates b and t cells, gives
memory for when the pathogen reappears
- Edward Jenner -> cowpox

Adaptive immunity

- Adaptive immune responses are mediated by lymphocytes of the lymphoid lineage

which fall into two major classes:

- T lymphocytes –
- kill virus-infected and cancerous cells (CD8+ T cells/cytotoxic T lymphocytes)
- activate other cells of the immune system (CD4+ helper T cells)

- B lymphocytes - secrete immunoglobulins

Natural killer (NK) cells – innate immunity

- The lymphoid lineage contains a third major cell type that lacks antigen-specific
receptors and is capable of killing virus-infected cells immediately without prior
activation and without clonal expansion
- These are known as natural killer (NK) cells
- They are generally regarded as part of the innate immune system (because they
don’t require prior activation)
- Activation occurs via binding of activating receptors to surface molecules produced
by cell damage e.g. cancer or that are encoded by infecting viruses
- All they do is they hunt and look for signs and abnormal antigens that are appearing
on surface of cells -> could be viruses splitting, where they kill them instantly
- Important for killing cancer cells or cells that are infected by viruses

The antigen - a definition

Any molecules or parts of molecules recognized by the
variable antigen receptors of lymphocytes are known
as antigens

Lymphoid lineage - cellular differentiation

- Mature lymphocytes that have not yet

encountered antigen are known as naïve
lymphocytes
- Differentiation into effector cells is stimulated
by encounter with antigen and is preceded with
a vigorous proliferation that selectively expands
the numbers of those lymphocytes with
receptors specific for the inducing antigen
- This is known as clonal selection of antigen-
specific lymphocytes

- Bone marrow Lymphoid progenitor -> Cytokine

Gear down and go down lymphoid pathway
- T cells -> into the thymus to grow up
(thymocyte are immature t cellls), then move
into blood and become mature but naïve helper
or cytotoxic t cells, havent bound to the antigen
yet. Migrate to secondary lympohoid tissue to
wait till dendritic cell to give them information.
- Dendritic cell can go down myeloid or lymphoid.
Mainly Myeloid mainly.
- Once B and T cell get activated by dendritic
cells, they will go into to the peripheral tissue
- B cells -> antibodies to cover pathogen for
opsonin for uptake by phagocyte
The Lymphatic System

- The critical interactions between activated dendritic cells and lymphocytes occurs in
lymphoid tissues
- Lymphocytes enter lymphoid tissues from the bloodstream
- Return to the bloodstream through the lymph if they are not activated
- Migration through lymphoid tissues, and cellular interactions, are guided and
controlled by adhesive cell surface molecules whose expression is regulated by
cytokines and chemokines
- Lymphoid tissues -> thymus, spleen, lymph nodes around the body, bone marrow

How do T lymphocytes recognise antigens?

 - T lymphocytes recognise antigens
by means of T cell receptors
generated during their
differentiation - they interact with
APCs (antigen-presenting cells) via
complementary ligands (Recognize
through APC (macrophage and
dendritic cell), using a t cell
receptor)
- Each individual lymphocyte
expresses an antigen receptor of
only one specificity, but the total
population of lymphocytes in any
given individual is collectively
capable of recognising virtually any antigen
- Massive population of t cell, each of which has its own specific receptor that only
recognises 1 antigen
- Only want 1 clonal lineage to be turned on at any one time
- Has a groove/variable region that will acept only that specific antigen

Clusters of Differentiation

The major classes of T cells are often referred to as CD4 and CD8 cells
• CD4 is a marker of helper T cells
• CD8 is a marker of cytotoxic T cells

Both are co-receptors for MHC molecules

• CD4 is a co-receptor for MHC class II molecules (Helper)
• CD8 is a co-receptor for MHC class I molecules (Killer)

These co-receptors play an important part in signal tranduction on antigen binding by T cells
Activation of the adaptive immune system

• Dendritic cells of the innate immune system play a crucial role in activating
adaptive immunity
• Begin life as phagocytes but when activated by conserved components of micro-
organisms, or by inflammatory cytokines released by macrophages, become
dedicated to displaying components of ingested micro-organisms for recognition
by naïve T lymphocytes and subsequent differentiation of naïve T lymphocytes
into effector T cells
• Dendritic cells -> -Phagocytosis -> recognises foreign -> relays to cells of the
adaptive immune system
• Effector t cells are activated t cells
Dendritic cells
• Especially numerous in epithelia and at mucosal surfaces
• Langerhans cells first recognized in the skin in the 19th century. They are as a subset
of dendritic cells that reside in the keratinised epidermis for several months
• Second population of skin-resident dendritic cells is found in the dermal layer -
known as dermal dendritic cells or interstitial dendritic cells
• In mucosal epithelium of the gut, dendritic cells are concentrated at specialized sites
of antigen collection that overlie lymphoid tissues
• Some have specialised surface properties that enable them to extend their long
dendritic processes between the cells of the epithelium and into the lumen to
sample antigens
• These cells are directly exposed to ingested antigens and to commensal bacteria as
well as invading pathogens
• These cells have a specialised role in preventing inflammatory responses to harmless
gut residents

Different types of dendritic cells

• Dendritic cells in the dermal layer of skin – dermal dendritic cells

• Dendritic cells in the epidermal layer of skin – langherhan cells
• M cells are specialised cells (not mast cells), samplying cells, take bit of food,
foreign antigens, transcytose them from the lumen of the gut feed them to
dendritic cells in the underlying tissue
• Some dendritic cells are stick out finger like projections through the gut
• Rest waiting for information below the gut may then have to move into
lymphatic system to activate adaptive immune system.
Activation of the adaptive immune system

• A dendritic cell displays a component of a micro-organism for recognition by

a T lymphocyte bearing a receptor for that component.
• This stimulates it to proliferate and differentiate into an effector cell.
• When the effector cell recognizes the same antigen displayed on the surface
of a B lymphocyte, the T cell activates the B lymphocyte, which in turn
proliferates and secretes antibodies that recognise the micro-organism

Dendritic cell migration and activation of t cells

• Dendritic cells the key APC of the immune system generated from pluripotent
cells in the bone marrow, migrate to peripheral tissue in the blood stream
• Dendritic cells have PRR that can recognize AMPs
• They can phagocytose the pathogens.
• When bound, they activate the dendritic cells and mature.
• Migrate from tissue and change their behavior to stop phagocytosis and to start
expressing immune stimulatory molecules
• Activated DC cells Migrate from the tissues to the lymphatic vessels
• The lymphatic fluid drains the lymph node, carrying the DC with it T cells, inspect
DC for the antigen
• T cells that do recognise specific antigen become activated and proliferate to
become effector cells
• T cell can mutate their receptor to get a perfect fit

T cell targeting

• Unlike B cells whose surface immunoglobulin molecules are adapted to serve

as antigen receptors, T cells are unable to interact directly with intact antigen
and so must interact with other cells of the immune system. B cell have a b cell
receptor on their outside where that is the antibody they will go on to produce
in large numbers
•

 How do T cells know which cells to interact with?

 • T cell only has a t cell receptor
• T cells are focused on their target cells by cell surface proteins known as MHC
molecules because they are encoded in the major histocompatibility complex
(MHC) - a region of the chromosome producing these things
• They interact with antigens that are expressed in the MHC class protein
complexes
• MHC molecules carry fragments of antigen, usually peptide fragments of
proteins, from internal compartments of the target cell to the cell surface and
display them for recognition by the T cell
• All naïve T cells are activated by antigen fragments displayed on MHC
molecules on the surface of dendritic cells, and after differentiation into
effector cells they are triggered to kill or activate cells displaying the same
complex of MHC and antigen

Peter Medawar (1944)

MHC compatibility for skin grafts, rejeceted, MHC complex must be matched with donor.

Major Histocompatibility Complex

 There are two classes of MHC molecules,

MHC class I molecules and MHC class II
molecules
 These monitor different internal
compartments of cells and are
recognized by distinct classes of T cells

MHC class 1 found on all cell surface of nucleated cells in the body
MHC class 2 normally found on APCs such as DC, phagocytes and B cells
Because neutrophils, with some exceptions (4, 5, 6), do not generally express MHC-
II molecules, their potential ability to function as APCs for CD4 T cell responses appears
limited.
Monitoring for infection

 The two internal compartments of cells that need to be monitored for pathogens
are:
1. The cytoplasm - All viruses and and some important bacterial pathogens replicate in
the cytoplasm
2. Vesicles of the endosomal/lysosomal pathway - Contain internalised antigens
derived from extracellular pathogens

In the specialised case of macrophages, vesicles may harbour bacteria and some fungi (e.g.
Cryptococcus) adapted to survive phagocytosis
 The cytoplasm is monitored by MHC class I molecules
  Vesicular compartments are monitored by MHC class II molecules

MHC class I molecules

 MHC class I molecules are expressed on virtually all body cells and bind peptides
generated by cytoplasmic proteases from cytoplasmic proteins
 Their critical role is to display antigens derived from pathogens that replicate in the
cytoplasm, and antigen displayed on MHC class I molecules is recognised by
cytotoxic T cells, which kill the infected cells
 This system ensures the destruction of cells in which cytoplasmic pathogens are
replicating and is believed to be essential for the elimination of some of these
pathogens
 MHC class 1 molecule Role is to take antigens from the cytoplasm and package them
up on MHC class 1 molecule and take them on the outside of the cell to display for
cytotoxic t cell.

Recognition of antigen and MHC class I molecules by cytotoxic T cells

Cytotoxic binds to MHC class 1 complex in the virus infected cells, once that docking
happens, the cd8 t cell release these 2 components and destinded for death.

 Peptide fragments of pathogens, such as viruses, that replicate in the cytoplasm

compartments of cells are carried to the cell surface by MHC class I molecules and
are recognised by cytotoxic T cells
 Upon activation, CD8 T cells produce:
 1. Perforins - proteins that form pores in the membrane of target cells
 2. Granzymes - proteases that induce programmed cell death on entry into target
cells

Cytotoxic T-cell killing

 Virus infect epithelial cells

 Degradation of virus proteins within the cell allow viral peptide to display to the cell
surface bound to MHC1 molecules
 CD8 t cells that recognize these MHC1 peptide complexes are activated and the kill
the infected cell
 Having kill 1 cell, it can move on to a new target kill that cell and move on
 Killing process when t cell receptor And CD8 together bind to a MHC class 1 molecule
baring a viral peptide, producing signals that activate the t cell. Cytotoxic t cells
 contain membrane vesicles called cytotoxic granules, which package the proteins
which kill target cells -> grazymes and perforin
 These proteins are complex with a scaffolding protein called serglycin
 Activation of the t cell cayuses the release of these vesicles deliverying granyzyems
and perforin or the target cell
 The perforin facilities the granyzumes into the cytosol
 At this point the target cell is destined for death and the t cell is moved on to find
another cell
 Process of cell death by the granzymes uses the same basic cellular machinary that
gives rise to apoptosis
 Granzymes target various cellular proteins that regulate apoptosis. BID and pro-
caspase 3
 Granzymes cleave BID, which in this truncated from, causes, cytochromc c to release
from the mitochondria to the cytosol
 Simultaneously, Granzyme B activates pro-caspase 3, which cleaves ICAD, the
inhibitor of the caspase activated DNAse, the activate DNAse migrates in to the
nucleus and degrades the DNA and ensuring cell death

MHC class II molecules

 MHC class II molecules are normally expressed only on cells of the immune system,
and in particular on B cells, macrophages and dendritic cells
 They bind to peptides generated from internalised antigen in the endosomal
compartments of cells (They bind to antigens produced during the phagocytic
process during the phagolysosome)
 Antigen derived from internalised micro-organisms by lysosomal proteases and
displayed by MHC class II molecules on dendritic cells is recognized by naïve T helper
cells at the initiation of immune responses

MHC Class II processing

 2 chains of the MHC class 2 molecule asembe in the endoplasmic reticulum

 With a portion of the 3rd chain invariant chain binds to the peptide binding groove of
the MHC class 2 and prevents peptides and unfolded proteins present in the ER from
binding.
 The invariant chain guides the transport of the class 2 molecule out of the ER
through the golgi apparatus into a vesicle that eventually becomes part of the
endocytotic pathway, by which apthogens and foreign protines are taken into the
cell.
  Progressive acidification, leads to MHC class 2 molecule being able to pick up
antigen, and take it to the outside of the cell to dispay to CD4 t cells
 Progressive acidfication of this endocytic vesicle activates proteases which cleave the
invariant chain in 2 places, leaving a small peptide, the CLIP, bound to the class 2
molecule. Engulfed pathogens are also degraded by acid activated proteases into
peptides, but these cannot bind to the MHC class 2 molecules as the CLIP peptide is
still bound to the class 2 moleule.
 Removal of the CLIP peptide is the function of HLADM, present in these vesicles,
functions as a catalyst, coordinating the release the CLIP peptide and the binding of
pathogen arrived peptides
 The MHC class 2 peptide complex is transported to the cell surface and then can be
recognised by antigen receptors of CD4 t cells.

Recognition of antigen and MHC class II molecules on dendritic cells by naïve helper T cells

 Naïve helper T cells are first activated when they recognize antigen bound by MHC
class II molecules on the surface of dendritic cells
 Dendritic cells taking up bacterium and microbial fragment, take it into the
endosomal pathway, fuses it with the compartment containing the MHC class 2
molecule, if the cell is stimulated recognizing it is an antigen, it will display it.
 Progressive acidification within these compartments allows the antigen to dock with
the groove on the MHC class 2 protein and taken out to display on the outside of the
dendritic cell
 The Naïve t cell moving through the lymphatic system looking for something that is
displayed by the dendritic cell that has moved from the peripheral tissues into the
lymphatic system. A t cell that has the correct receptor for that antigen displayed in
the MHC class 2 will move from being a naïve t cell into a effector cell. Its job is then
to move around search for cells within the peripheral tissues that is displaying the
same antigen.
 DC cell relay to CD4 t cell, cd4 t cell goes through clonal expansion, migrates out of
the lymphatic system into the peripheral tissues to help the macrophages dealing
with the lymphatic system

Recognition of antigen and MHC class II molecules on macrophages by helper T cells

 Helper T cells recognise fragments of a pathogen that has been internalized by

macrophages and have undergone proteolytic digestion to generate fragments that
are carried to the cell surface by MHC class II molecules.
 The helper cell is then stimulated to activate the macrophage to destroy the
internalised pathogen.
 This is particularly important for defense against bacteria that are able to grow in the
internal vesicles of macrophages
  Info sent down to find that cell displaying the particular antigen
 Macrophage peripheral tissues taking in the pathogen and destroying it within the
phagolysosome at the same time, it is taking bits of information and exposing it on
its cell surface via the MHC class 2 complex
 CD4 t cell migrated having received information from DC cell into the peripheral
tissues, finds it, docks with t cell receptor with the MHC class 2 complex with the
antigen, CD4 binds, stimulates the CD4 t cell to send a chemical molecule out to the
tell the macrophage to kill it.
 Macrophages are good engulfing things, display parts of it, CD4 comes in and helps
release cytokine to help the macrophage kill it

Recognition of antigen and MHC class II molecules on B cells by helper T cells

 B cells internalise antigens bound to their surface immunoglobulin and deliver them
to the endosomal compartments of the cell where they are digested into fragments.
 The antigen fragments then bind to MHC class II molecules which carry them to the
cell surface where they are recognised by helper T cells, which then activate the B
cell to proliferate and differentiate into an antibody-secreting plasma cell

 B cell receptor -> antibody it will produce

 Binds to the antigen on the outside of the viral particle, take that into the cell and
the MHC class 2 complex will take that antigen from that antibody and display it on a
MHC class 2 complex molecule and a CD4 T cell bind to it to stimulate the B cell to go
through massive proliferation to produce a massive number of the same antibody
CD4+ T cell sub set

Th1 has its own own cytokine profile, geared

up to produce cytokines to do specific things
-> activate macrophages
Th2 -> Activates B cells to produce
antibodies and inhibits macrophages

Co-stimulator pair activation

 Don’t just have CD4 and CD8, many array of costimulator pair that are bought in and
increase stimulation of immune response
 APC express MHC2, co-stimulator molecules such as CD80
 Stimulator of a cd4 t cell, by the APC, involves the interaction of the t cell receptor
and co-receptor molecules of the MHC peptide complex, as well as the interaction
between CD80 and CD28 (on the CD4 t cell)
 Interaction between APC and T cell cause signals to pass in both directions signaling
the APC to produce additionally co-stimulatory molecules such as CD86 and CD40,
signals through the t cell receptor and CD28 induce the t cell to produce CD40 ligand.
Interaction between CD40 and CD40 ligand as well as additional stimulation through
CD28 mediated with interaction with CD86 result in the full activation of the CD4 t
cell.
 Activation of CD8 t cells also require multiple receptor ligand interactions. The same
activation signals induce the APC to express other costimulaotry molecules such as
4-1BBL. While the initial activation of CD8 t cells induces the express of 4-1BB.
Binding of 4-1bbL and 4-1bb is though to required for full activation of CD8 t cell.
 Don’t have to know all.
 Starts with CD8 and CD4, if the fit gets stronger, there will be more docking
stimulating the immune interaction. If it is weak, there is no other co-stimulator
pairs produced and the t cell will move off. If it is a good fit, other ligands will come
into play.
Summary of Immune Response

 Immune response involves events that unfold both locally at the site of the infection
and distant sites such as nearby lymph nodes.
 Most pathogens kept outside of the body by epithelial barriers such as epidermis.
Only crossed when there is injury or tissue damage.
 After infection, bacteria cross the epidermis and establishes an infection in the
underlying tissue. Phagocytic cells in the tissue such as macrophages and neutrophils
engulf the pathogen. Dendritic cells are also phagocytic and are activated by binding
pathogens to leave the site of infection and migrate to a lymph node.
 It enters the lymphatic vessels and are collected by a draining lymph node. In the
lymph node, t cells are activated by antigens presenting by the dendritic cells, and in
turn activate b cells to secrete antibody.
 Effector t cells and antibody molecules return to the circulation. Leave the
circulation at the site of infection where inflammatory mediators have induced
changes in the blood vessel endothelium. CD4 t cells activate macrophages to
become more cytotoxic while antibody recruits complement to lyse bacteria directly
and to opsonize them to enhance their uptake by phagocytes.
 In the case of viral infections, activated CD8 t cells will kill any infected cells present.
Lecture 5 – Antibody Molecules

What are they?

• Host proteins produced in response to the presence of foreign molecules in the
body
• Synthesised primarily by plasma cells (lymphoid lineage) and are components of the
adaptive immune system
• Circulate throughout the blood and lymph where they bind to foreign antigens
• Antibody-antigen complexes are removed from circulation primarily through
phagocytosis by macrophages

 Plasma cells (b cell effector cells)

 Soluble
 Move about till they find the antigens
 Oposonize cells, make the cell bigger for phagocytic cell to regonzize pathogen

Linus Pauling - Proposed a single immnunoprotein reacted with all antigens, but its
conformation was different in each case (Wrong)

Frank Macfarlane Burnet

 Challenge pauling
 New theory
 Clonal selection theory
 As you developed you have antibody producing b cells but it gone through somatic
mutation during embroynic life
 As that development process occurred, it eliminated cells that was going to kill you
Clonal Selection of Antigen-Specific Lymphocytes

 As you develop you have a pool of immature lymphocytes,

you go through clonal deletion for self tolerance
 As you develop b cells produce antibodies against self
antigens are tested, and if they bound to self antigens they
are eliminated from the immune system so only left with b
cells with capacity that can bound with foreign antigens
 Self-tolerance occurs through clonal deletion
 Larger pool -> reduce the self harming one
 When 1 or a number of b cells challenged and feed
information through immune system and a cd4 t cell
comes along and test it and says it’s a good feed and it
stimulates, there will be clonal expansion.
 Single b cell producing antibody you want. When infection
dealth with, they sit back and become memory b cells and
wait till the same infection comes.
Induction of B cell Antibody Response by T cells

 Germinal centers where b cells proliferate and undergo isotype switching and
somatic hypermutation.
 The formation of germinal center start when dendritic cell present antigens on their
surface, activating antigen speicifc cd4 t cells, which proliferate and become effector
cells which are capable of activating antigen speciifc b cells.
 Once activated by a cd4 t cell, the b cell prolioferates, to form a primary focus of
antigen specific b cell. B cell from the primary focus migrate to nearby follicles and
proliferate, other b cells from the primary focus persist in the t cell area for a short
while and secrete the antibody but eventually die
 B cell that enter the follicle begin to proliferate rapidly. They also go through somatic
mutation to introduce new variable into the b cell receptor. B cell go through a
selection after somatic mutation where the receptors are tested if they can bind to
the antigen
 If they fail to bind or don’t bind efficacy, they die.

 Somatic hypermutation is a cellular mechanism by which the immune system adapts

to the new foreign elements that confront it
 Immunoglobulin class switching, isotype switching, is a biological mechanism that
changes a B cell's production of immunoglobulin from one type to another, such as
from the isotype IgM to the isotype IgG.

Antibody Structure

 Large family of glycoproteins

 Large polypeptides that have bits of carbohydrate on them.
 Share key structural and functional features
  Functional - able to bind both to antigens and to specialised cells or proteins of the
immune system
 Structural - composed of one or more copies of a characteristic unit that form a Y
shape

Antigens, Epitopes, Paratopes

 Antibodies bind to them

 Any molecules or parts of molecules recognised by the variable antigen receptors of
lymphocytes are known as antigens
 The epitope or antigenic determinant is the specific region of the antigen bound by
the variable region of an immunoglobulin
 The paratope is the antigen-binding region of an antibody
 Amino acid sequence on a polypeptide, co-protein on the outside of the virus for
example
 Don’t recognize the whole protein, they recognize the specific amino acid sequence
up to 5 amino acids in length
 If you change 1 of the amino acids, the antibody will lose the ability to bind to the
antigen.
Affinity

 Affinity is the measure of the strength of the binding of an antigen by an antibody

 Antibody binding to antigen is non-covalent and reversible
 The affinity of an antibody (Ab) for an antigen (Ag) is related to the ratio of the rates
of the forward reaction for formation of the complex to back reaction for decay of
the complex

 Antibodies don’t come along and bind

 They bind in a specific formula
 Move backward and forward determine if another antibody that comes along that
has a better fit.
 If somatic hypermutation, and has a better affinity antibody, the first antibody lifts
off and the better match is bound to the antigen.

Antibody structure
Not rigid structures, can contort itself to bring in the fab domain to recognize the amino acid
sequences

2 heavy (higher molecular weigtt) and 2 light chains make up basic y shape
2 light chains on the antigen binding site end that do the binding
Each Y contains four polypeptides
• Two identical heavy chains (55kDa
each)
• Two identical light chains (22kDa
each)
• The four polypeptide chains are held
together by disulphide bridges and
non-covalent bonds
• The two domains that carry the
antigen binding sites are known as Fab
fragments (fragment having the
antigen binding site)
• The third domain is known as the Fc fragment (the
fragment that crystallizes)

Top bit is the one that binds to the antigen (has antigen binding site) Fab
domain. Fragment having the antigen binding site
Fc domain - effector part of the antibody, speaks to the cell of the immune system
or deals with compliment.
Basic Structure of an Antibody Molecule

 The ability to bind antigen is

determined by the N-terminal
domains. These differ
considerably between
antibodies of differing
specificities and are therefore
termed ‘variable’ or ‘V’
domains
 VH + VL is the antibody
binding site, gives antibodies
the diversity
 Variablility happens at the N
terminus
 CL + CH constant light and constant heavy
Hyper-variability

 Sequence analysis of amino

acids of VH and VL regions
reveals small regions of hyper-
variability within four
conserved framework regions
(FR1, FR2, FR3 and FR4)
 In the 3-dimensional structure,
the hyper-variable regions
form loops that combine
together to form the principal
antigen-binding surfaces
 These surfaces are called the complementarity determining regions or CDRs (CDR1,
CDR2 and CDR3)

There are Loops within the fab domain, loops determine the hypervariability within the
antibody structure

Have framework regions dispersed within that variable region.

CDRs that give the antibody the specific capability and binding ability to a speciifc antigen.

Rodney Porter (1959) & Gerald Edelman (1961)

 Investigation of antibody structure
 Clonal expansion and clonal structure
 Looking at single IgG
 Using different enzymes to degrade antibody into their constituents parts
 They have 3 parts - 1 and 2 bound to antigen without forming a precipitate , 3 could
be crystallised
Proteolytic Fragmentation of Antibodies

Using papain -> protease which breaks the antibody into 3 different parts
Fab, and intact Fc
Pepsin cleavage
Fab2 and fc fragments
TO make smaller fragments to look at function

Classes of Immunoglobulin

• There are five main classes of antibody in serum

IgG, IgM, IgA, IgE and IgD
• The five classes are divided on the basis of the number of Y-like units and the type
of heavy chain polypeptide they contain (g, m, a, e and d)
• While there are five different types of heavy chains, there are only two light chains
(k or l)
• One light chain always associates with one heavy chain, so the number of light
chains always equals the number of heavy chains

Immunoglobulin Sub-Classes

In humans, the IgG class has four sub-classes

IgG1, IgG2, IgG3 and IgG4
These have g1, g2, g3 and g4 heavy chains
The IgA class has two sub-classes
IgA1 and IgA2
These have a1 and a2 chains respectively
Know the formula

 IgM primary response, low affinity, not brilliant fit, IgM

 Mutation of B cell receptors, switch into IgG -> secondary response, principle IgG in
the blood
 IgA protect mucous membrane
 IgE parasites
 IgD no idea
J chains and Secretory
Components

 IgM Held together by

something called a J
chain (pentamer)
 IgG single unit
 IgA dimer -> also have
a J Chain, and also a
secretory component,
move out of blood
stream across barrior
and sit on mucosal
lining to deal with
pathogens

Movement of Antibodies across Mucosal Barriers

 Gut
 M cells -> sampling cells relaying them to the DC cells underneath that feed
information to the rest of the immune system
 If a Dc cell has gone to talk to cd4 t cell and then gone to B cell and said we need
your antibody, the antibody has moved out from B cell into the gut where it can deal
with the antigen and pathogen that has got to be dealt with.
 B cell can’t do that, can only secrete anybody, it has to move around the intestinal
wall to get to the bacterium that has tried to invade the gut.
 Does this by the dimer called the secretary component (IgA)

Secretory IgA

 IgA is the most prevalent immunoglobulin in mucosal secretions

 Binds to antigens and prevents the uptake of bacteria and toxins
 Epithelial cells in the intestine mediate the transfer of dimeric serum IgA to the
intestinal lumen via the polymeric Ig receptor
 It binds to the secretary component, the antibody is then moved out across the wall
and into the gut
Transepithelial transport of IgA

 IgA dimer with J chain attached binds to the polymeric Ig receptor, a transmembrane
protein expressed at the basal surface of epithelial cells of the gut, airways and
various secretory glands
 Binding of IgA induces transcytosis of the polymeric Ig receptor, this stimulates the
cell to take the antibody up and transcytose it across to the luminal side of the gut to
deal with the bacterium
 Complex is delivered to the apical surface of the epithelial cell and into the lumen
 Proteases cleave the pIgR near the membrane, releasing the majority of the
extracellular domain still bound to IgA dimer
 Polymeric Ig receptor protects IgA against protesase produced by bacteria to break
down antibody
 The ‘secretory component’ protects IgA from proteases present in mucus and
anchors IgA at the desired location
 When get to the other side, the IgA will opsonize the bacteria, complex it, take its
toxins and complex them, phagocytic cells will come along bind to Fc domain of the
antibody that is sticking out and the whole thing will be destroyed.

Opsonisation and Phagocytosis

 After opsonisation with IgA, the receptor Fcalpha/mewR mediates the uptake of
organisms by macrophages and dendritic cells
 A second receptor FcaRI mediates uptake by neutrophils
 IgA deficiency is a common immunodeficiency in humans
 IgM can compensate for IgA deficiency by also binding to pIgR
 IgM opsonised organisms are phagocytosed via Fca/mR
 Activating Fc receptors signal through ITAMs

ITAMS

 Antigen receptors, Fc receptors and some of the activating receptors of natural killer
cells signal via a common mechanism that is dependent on a conserved amino-acid
sequence motif
 This is known as the immunoreceptor tyrosine-based activation motif
 The ITAM contains two precisely spaced tyrosines within a consensus sequence
 When phosphorylated, the tyrosine residues provide a binding site for one or two
closely related intracellular tyrosine kinases
 Syk in most immune cells
 ZAP-70 in T cells
 These have tandem SH2 (Src Homology 2) domains spaced at exactly the right
distance apart to dock onto the two phosphotyrosines, and that activate signaling
events downstream of the receptor
Signal Transduction by ITAM-containing Immunoreceptors

 The surface immunoglobulin (Ig) that serves as the B cell receptor for antigen brings
the receptor into proximity with a Src-family tyrosine kinase, which is held in the
plasma membrane by a lipid tail and phosphorylates ITAM tyrosines in the receptor
Iga and Igb chains
 This creates a binding site for Syk, which binds to the doubly phosphorylated ITAM
sequence
 Once bound, Syk becomes activated by tyrosine phosphorylation and it then
phosphorylates other signaling proteins that ultimately bring about changes in the
behaviour of the cell e.g. antibody production

Opsonisation and Phagocytosis

 After opsonisation is outside.

 Uptake of bacteria by phagocyte is an active process which requires the triggering of
specific recpetors on the phagocyte, Fc receptors. Which bind antibody hooded
bacteria is capable of triggering phagocytosis. Binding the antibody molecules on the
Fc receptor on the phagocyte causes the cell to engulf the bacteria.
 The phagocyte produce ruffles or psydopods, that surround the bacteria and fuse,
trapping the bacteria in a intracellular vesicle, a phagosome. Within the phagocyte,
lysosome fuse with the phagosome, delivering the content to degrade the engulfed
bacteria

Antibody-Mediated Cytotoxicity
 The two most widely recognised mechanisms of antibody- mediated toxicity are:
1. Antibody-dependent cell-mediated cytotoxicity (ADCC)
Antibody attracts cytotoxic cells by means of their Fc receptors. Antibody binds, phagocytic
cells kill whatever the antibody binds to.

2. Complement-mediated cytotoxicity (CMC)

Antibody binding results in the fixation of complement onto the target cell. Antibody fixes
complement of the outside of the cell to kill it.

 Fc receptor binds (once opsonized), forced the eosinophil to degranule in the

proximity of the parasite and make life horrible for the parasite.
 Phagocytosis by a macrophage, opsinsation of bacterium, destruction in
phagolysosome.
 IgE, bind to antigen, degranulate, IgE once opsined, binds to FcEpsilon. Make the cell
degranulate the toxic components.
 Basophils, eosinophils and mast cells have a high affinity of IgE, which release
histmaines etc.

Fc receptors
  FCeR1 -> mast cells, basophils, eosinophils, for degranulation (histamines)
(eosinophil no histamines)
Complement
The complement system, also known as complement, consists of about 30 serum and
membrane proteins that can mediate a variety of immune reactions including:
• Triggering inflammatory responses
• Attraction of phagocytes to sites of inflammation
• Degradation of membranes or virus envelopes
• Stimulation of antibody production

Complement, within your serum you have humoral components (soluble components).
Have 30 proteins that from different components of the complement system

Activation of Complement
The active components of complement are generated from inactive precursors by a cascade
of proteolytic reactions
These are triggered through 3 different pathways:

The LECTIN pathway (recognition of carbohydrate moieties e.g. by collectins such as

mannose-binding lectin)
Lectin – recognises carbohydrates structures by things such as collectins such as mannose
binding lectin

The CLASSICAL pathway (antibody binding to antigen in immune complexes)

Classical – antibody based, antibody binding to antigen in immune complexes, then
stimulates complement on the cell surface where the antibody is bound

The ALTERNATIVE pathway (directly at microbial cell surfaces)

Alternative – positive loop, keeps depositing component of complement on cell surface

Activation of complement by any one of the pathways initiates a cascade of cleavages in

which each component in the pathway is cleaved into two fragments:
1. The small fragment (a fragment)
2. The big fragment (b fragment)
The b fragment forms a subunit of the protease complex mediating the next cleavage in the
pathway
The reactions can be divided into early events, in which the components are not the same
for the same three pathways and late events, in which they are identical

Start up differently, but end up in the same place.

Early and Late events

 The function of the early events is to generate two functionally equivalent forms of a
protease, known as C3 convertase
 C3 convertase then initiates the late events to produce the effector components of
complement
 C3 convertase covalently attaches to the cell surface at which the complement
activation was initiated
 Cleavage of complement component C3 leads to effector activation
 In this way, the effects of complement activation are confined to the infectious
organism that triggered

Classical pathway
Pentraxins = Pentameric serum proteins that participate in innate immunity and can also
activate the Classical pathway of complement

-activated by pentraxin (primitive antibody in mammals) or antigen-antibody complex

Alternative pathway -> amlification loop

MASP - Mannose-binding, lectin-Associated,
Serine Protease

Mannose binding lectin binds to mannose on

the outside of the cell, you have proteases on
the legs known as MASPs, they activated, end
with proteolytic cascade which ends up into C3
convertase.

Collectins & Ficolins

 The epithelium of the respiratory tract is lubricated by a layer of phospholipids and

proteins known as surfactants
 Two of these, Surfactant Protein-A (SP-A) and Surfactant Protein-D (SP-D) are
Collectins and function as opsonins, coating microorganisms (e.g. Pneumocystis in
AIDs patients - Lecture 8) and stimulating uptake by phagocytes
 The Collectin family also includes Mannose-Binding Lectin (MBL) - binds to mannose-
containing carbohydrates on surface of viruses, bacteria and fungi
 Ficolins bind to N-acetylglucosamine

 Leptin pathway
 Lungs have surfactants, gives spits throth, bit like washing up liquid on your lungs
 Surfactant protein A and Surfactant protein D combined are known as collectins.
 Good at coating microorganisms and stimulating uptake by phagocytosis.
 Collectin family also includes mannose binding lectin, to bind to mannose, sugar
molecules, containing carbohydrates on surfaces of pathogens.
  Ficolins bind to N-acetylglucosamine, sugar found in pathogens, not in humans
 these swtich on leptin pathway

 Each polypeptide chain of the collectin family of

 proteins consists of an amino-terminal cysteine-rich region
followed by a collagen-like region, an a-helical neck region and a
carboxy-terminal globular domain (a C-type lectin known as the
carbohydrate recognition domain [CRD])
 The polypeptide chains trimerise
 Both MBL and SP-A are hexameric structures. The multiple arrays
of CRDs give high affinity binding to polysaccharide ligands in
microbial cell walls
 Particle recognition is translated into immune action:
1. By phagocytes, which are stimulated to internalize bound
microorganisms or,
2. By conformational changes in the collectin that activate
pre-bound protease subunits and initiate the complement cascade

Membrane Attack Complex

C3b + C3 convertase → C5 convertase

C5 convertase + C5 → C5a + C5b
C5b + C6 + C7 + C8 + C9 ⇢ MAC
 ☠︎

C3a is a inflammatory mediator important in stimulating inflammation and recruiting cells to

the site of the infection
C3b binds to C3 convertase makes C5 convertase.
C5 convertase + C5 -> C5a and C5b
C5b, big cascade events, in strict order, called a MAC
Membrane attack complex, which punches holes in the outside of the wall of the virus or
bacteria. C9 does the punching holes on the outside of cell wall.
Lecture 6 – bacterial Immunity
Innate rapid – hours days, broad, low level protection
Adaptive – slow, days, specific, high level protection
Intimately linked

Bacteria & The Innate System

The innate system recognises molecules that are common to bacteria but absent from the
host

 Pathogen-Associated Molecular Patterns (PAMPs)

 The innate system is pre-coded to recognise a limited repertoire of molecules
 This enables it to respond quickly, but only to provide a low level response

Way in which recognition events trigger 2 important pathway that protect against disease

• Killing by complement
• Phagocyte killing

Complement plays a role in protection against microorganisms

  Best studied are roles in protection against bacteria
 Also active towards fungi, viruses and protozoa

3 pathways of complement activation

In all pathways, the first step is the recognition of microbial components

After recognition – early events

 Early events generate a protease, known as C3 convertase

 C3 convertase covalently attaches to the cell surface at which the complement
activation was initiated
 In this way, the effects of complement activation are confined to the infectious
organism

 C3a and C3b tethered to the surface of

microorganism
 Response is localized to that
microorganism
 C3 convertase breaks c3 into c3a and c3b

After recognition – late events

  C3b thethered to the surface of the microorgniasm, forms a key component of c5
convertase
 Converts C5 into c5a and c5b
 C5b will form MAC along with c6-C9

Protective mechanisms of complement

C3a -> release when c3 is cleaved by the c3 convertase, promotes inflammation

C3b -> tethered to the surface when c3 is cleaved, part of the c5 convertase complex, and
also does opsonization and phagocytosis
C5a -> promotes inflammation
C5b-C9 -> lysis of the microbe

Action of C3a and C5a

  Soluble molecules that promote inflammation.
 Bind to mast cells via c3a and c5a receptors, cause degranulation of mast cells and
release of histamines.
 Histamines promotes vasodilation, increases blood supply to the site of infection,
allows other element so of the immune system to the site of inflammation.
 They also act as chemoattractants
 C5a in particular, signals to phagocytes to go towards site of attraction
 C5a generated at the site of infection signals the phagocytes to move towards site of
attraction

Membrane attack complex forms a pore in the target membrane

 C9 forms a pore, has a hole down in the middle, inserts in the bacterial membrnane
and punches a hole in the membrane, and kills the bacteria promotes unregulated
ions across the membrane.
 End of the cascade, form MAC
 C5b,C6-C9

Complement deficiency and disease

C1q – meningitis by Neisseria, respiratory tract infection by streptococcus pneumoniae

C3 - meningitis by Neisseria, respiratory tract infection by streptococcus pneumoniae
C5 - meningitis by Neisseria
C6 - meningitis by Neisseria

Bacterial recognition by phagocytes

Indirect recognition
Bacteria coated with antibodies are recognised by antibody receptors (e.g. Fc receptors)
Bacteria coated with C3b are recognised by C3b receptors

Direct Recognition
Pathogen Associated Molecular Patterns
RECOGNISED BY
Pattern Recognition Receptors , eg.
Toll-like receptors (TLRs)

Dimerisation is essential for signalling

 PRR
 Imbedded in the surface of the phagocytic cell
 Dimer of the toll like receptor
 Horse shoe shape on the outside of TLR does recognition
 Recognize different structures on the surface of bacteria

TLR ligand source

TLR1/2 Triacyl lipopeptides, peptidoglycan Bacteria, mycoplasma

Diacyl lipopeptides, lipoteichoic
TLR2/6 Bacteria, fungi
acid, zymosan
TLR3 Ds RNA viruses

TLR4 Lipopolysaccharide bacteria

TLR5 Flagellin bacteria

TLR6 Diacyl lipoprotein Bacteria

TLR7 ssRNA Viruses

TLR8 ssRNA Viruses

TLR9 CpG DNA Bacteria, Viruses

TLR10 NK

TLR11† Profilin Protozoa

TLR12† Profilin Protozoa

TLR13† 23S rRNA Bacteria

Consequences of binding to phagocytes

• Activation of pathways leading to cytokine and chemokine production

• Ingestion and killing

TLR signalling and inflammatory responses

 Recognition of bacterial molecule by TLR -> Dimerisation of the TLR -> signalling and
inflammatory responses
 MyD88 and TRIF pathway
 End with cytokines

Actions of cytokines

Cytokine action

Bacteria recognised by TLRs are ingested

IL-1 Activates endothelial
and (killed) by phagocytes
cells, fever
Killing in the phagosome
IL-6 Proliferation of
 Range of different chemicals antibody-producing B-
thrown in the phagosome to kill cells
the microorganism.
TNFα Activates endothelial
 Oxygen radicals, lower pH (acidic,
cells, fever
ph3-4), enzymes (lysozyme,
Activates neutrophils
protease)
 Few bacteria can escpae the
phagosome and avoid killing

interferon α Antiviral immunity

Standard therapy for Promote CD4+ and
hepatitis C virus and CD8+ T-cell responses
hepatitis B virus
infections
Bacteria & The Adaptive System

The adaptive system recognises antigens (motifs) that are unique to an individual pathogen

It takes time to develop and mature, but can provides a very powerful protective response

Most common types of bacterial antigen recognised by the adaptive system

• Proteins
• Polysaccharides

On the surface of the bacterial cell

Recognised by the adaptive system
In particular proteins are important for protection against disease

Bacterial proteins are processed by antigen presenting cells

 Bacterium taken in a phagosome. Most cant survive in the phagosome after all the
chemicals throw in. Some bacteria can escape into the cytosol, and can avoid killing
mechanism.
 These different pathways dictate what type of adaptive reseponse develops
 If bacteria get taken up into a phagocyte into a killing pathway, phagolysosome, or if
it escapes into the cytosol, it has 2 different responses.
 Presentation of bacteria by phagocytes or other APC to the immune system.
MHC Presentational pathways

Class 1 – CD8 t
cells, protein
within the cytosol

Class 2 – CD4 t
cells, protein taken
up into the
phagosome

MHC1 on all nucleated cells

MHC2 on APCs

Top bacteria escape. MHC class 1. CD8+.

Protein recognized in the cytosol, processed by the proteasome, complexed with MHC class
1, and eventually, peptides derived from the proteins become presented in MHC 1 on the
surface of the cell. Peptides eventually recognized by cytotoxic cd8 t cells.

CD8 t cells activated, effective at killing infected cells. Instead of phagocyte killing the cell,
the immune system kills the infected cell. Release enzymes such as perforin, granzymes that
punch holes in the membrane.

• Diseases where the bacteria invade and grow within host cells
• tuberculosis
• salmonellosis
• listeriosis
• melioidosis
Bottom Bacteria in the phagosome.
Cant escape the phagosome, get killed by the phagosome.
Protein and peptides in the phagolysosome are processed in another pathway. Peptides
complexed with MHC class 2, presenting on the surface of the cell. Trigger CD4+ helper t
cell.

CD8 about killing

CD4 is switching immune system on to kill bacteria
Release cytokines to activate inflammation, antibody response, macrophage activation,
phagocytosis and bacterial killing
Take up more bacteria -> coat bacteria with antibody

Summary
Bacteria & antibodies

Protective mechanisms of antibodies – complement binding

• Antibodies bound to bacteria promote complement binding and complement

activation by the classical pathway
• After activating the complement pathway, they have all these effects promoted

Protective mechanisms of antibodies,

aggluntinated, neutralisation, opsonisation

• Agglutinated bacteria are cleared

more efficiently by phagocytes
• If toxins are central to the disease
process then antibodies that
neutralise the toxin block disease
• Antibodies bound to bacteria
promote their uptake and killing by
phagocytes

Diphtheria antitoxin – 1891 onwards

• Antibody to toxin raised in horses
• serum used to treat disease

Bacterial diseases where antibody plays a key role in protection

 Tetanus – antibody neutraluzes toxin
 Streoticiccal pneumonia – antibody aggulinnates, opsonises, complement activating
 Meningitis – antibody
  Anthrax – antibody neutralise toxin
 PLague
Lecture 7 – immune responses to fungal and parasitic infections

Fungal – oppurtunistic
Parasitic - everywhere

Fungi and parasites

Comprise a large group of genetically complex organisms capable of infecting humans

The immune system responds by organising specific types of innate and adaptive host
immune cells, which act together to mediate host defence

Evolution has resulted in adaptation by many of these types of organisms, resulting in

chronic or recurrent infections that can last many years

Fungi
Unicellular (yeasts) and multicellular (filamentous) organisms

Ubiquitous components of terrestrial environments where they contribute to nutrient

cycling

Some, termed endemic mycoses, reside in geographic areas with distinct climatic conditions
Many fungi dispersed by air-borne spores that can reach terminal airspaces in the lung
(Aspergillus fumigatus)

Although many are transmitted from the environment, fungi that cause disease can also be
part of the normal commensal microbial flora of humans, such as Candida albicans (in gut)

Mycoses – fungal infection

Candida albicans, Cryptoccocus, Aspergillus,

Rate of mortality is really high

Infection is not that high
Cure fungal infection is to get their immunity back up.
Candida albicans

A commensal fungus that colonises the normal human oral, gastrointestinal and urogenital
mucosa

Disruption of mucosal barriers leads to persistent superficial infection

Thrush, a superficial infection of the oral mucosa, and Candida esophagitis are common
opportunistic infections in patients with HIV
In the setting of compromised immunity, infections can disseminate to cause life-
threatening infections of virtually any organ
Candida is the most common infection of humans. In the USA, it is the 4 th most common
nosocomial bloodstream infection, and adds over $1 billion per year in medical costs

Viruence

Candida reproduces asexually by budding

In response to microenvironmental signals that accompany tissue invasion (changes in pH,
cell density, exposure to serum and iron deprivation), the fungus switches from the yeast
form to filamentous form.

The ability to switch morphologies has been linked to pathogenicity

Both forms are likely to contribute to virulence:

• Yeast form more easily disseminated through bloodstream
• Filamentous form facilitates invasion and evasion of phagocytosis

Mutants unable to make the dimporphic switch are commonly non-pathogenic in animal
models

once it breaks through the muscosal barrier is that is dimorphic

It has a yeast phase (budding phase), senses it has gone in blood stream, change in ph,
exposed to serum, iron depleted, and it switches from yeast phase to produce invasive
hyphae.

Mutants that lost dymorphic switch is not pathogenic.

When it gets in the blood stream, it switches to hyphae growth which causes major damage
Host recognition of fungal infection

Innate recognition of fungi is mediated by interactions with conserved

cell wall constituents (glucans, mannans, galactans), shedding of the cell as it divides as a
budding yeast or hyphae

Dectin-1 is highly conserved on the surface of dendritic cells and in lesser amounts on
macrophages

It has a C-type lectin carbohydrate recognition domain that mediates

the recognition of b1,3- and b1,6-linked glucans on the surface of intact cells and zymosan
(cell wall fragments consisting of b-glucans, mannans, mannoproteins and chitin)

Engagement of Dectin-1 results in tyrosine phosporylation of its cytoplasmic

immunoreceptor tyrosine-based activation motif (ITAM)

Dectin-1 acts synergistically with TLRs to induce production of inflammatory cytokines and
the production of reactive oxygen species (ROS)

These PRR don’t work in isolation, they work synergistically with other PRR in particularly
TLR. Synergistic siganlling increases the signal within the cell to tell it to go in the killing
process, using the ROS. Signalling down stream causes ROS to kill fungus.

PRR can recognise Structures of the fungal cell wall.

Chitin, beta1,6 and 1,3 glucan, mannan and galactan, ergosterol, glucan synthase

Different PRRs.
Dectin 1 binds to fungi, signals through a ITAM like motif phosphorylation with a SRC kinase
which sends the information into the cell.

Dectin 2 – PRR outside on innate cells, recognise fungi and eggs of parasites.

Mincle – Signals through ITAM, leads to innate inflammation, switch on adaptive immunity

Starts innate inflammation and leads to adaptive immunity.

Dectin-1 plays a non-redundant role in anti-fungal immunity

Knockout Dectin 1 receptor, challenge them with candida, those with dectin 1, resistant to
challenge, those who have double knockout die very fast. Same to aspegillus furmigatus.
Toll-Like Receptors

• Dendritic cells and other immune cells must be able to distinguish between
apoptotic particles generated by normal tissue turnover and particles that are
indicative of infection
• The molecules mostly responsible for making this pivotal distinction are those of the
family of Toll-like receptors (TLRs)
• Stimulation of immune cells through their TLRs leads to synthesis and secretion of
proinflammatory cytokines, thereby initiating the inflammatory response that
recruits both soluble immune components and immune cells from the blood
• TLR stimulation of dendritic cells also induces the initiation of an adaptive immune
response

Toll-like receptor activation

• Innate immune system recognizes pathogens and provides first line of defence.
• DC cells circulating through the tissues recognize PAMPS. PAMPs features of
pathogens such as LPS.
• DC cells recognise PAMPS through TLRs. In the case of LPS, it is recognized by TLR4
expressed on the surface on DC cells.
• LPS is transported by the soluble LPS binding protein LBP to the surface on the DC
cell and deposited on the cell surface protein CD14. the presence of LPS is detected
by TLR4 through the interaction and recognition of the LPS bound to CD14. The
signal delivered by the TLR4, causes maturation of DC cells, which travels to the
regional lymph node and activate the aquired immune response.

TLRs and their ligands

• Most known TLRs signal through MyD88 (Myeloid Differentiation factor 88). MyD88
is an adaptor protein that binds to TLR following activation by fungal cell wall
components and CD14.
• This initiates a signalling cascade that culminates in the transcriptional activation of
cytokine genes by NF-B
• NF-B (nuclear factor B): any of a small family of dimeric DNA-binding proteins that
mostly function as transcriptional activators and have a central role in both innate
and adaptive immune responses

Bacteria is TLR4
Fungi, TLR2 – cell wall components, also using CD14, adaptive protein for beta 1-6 glucan
and beta 1-3 glucan that interacts with that and docks with TLR2, adapter protein MyD88
once that is activated, there is activation of the NfKb and leads to transcription of the pro-
inflamamotry cytokines.
TLR2 is most abundant on macrophages and DC cells.
These cells doing the innate recognition of the different components of the fungal cell.

Collaborative signalling for cytokine production

• TLR binding to cell surface component and also Dectin 1 binding

• Get synergestic interaction where there is increased signal and enhanced cytokine
and chemokine production within the cell, where they speak to other immune cells
and recruit them to the site of infection
• TLR relies on adapter protein MyD88
• Dectin-1 signalling through ITAM with a SRC kinase that is phosphorylating the
motifs leading to signal transduction

Mannose-binding lectin and complement

Candida expresses surface adhesins and mannoproteins that activate the alternative and
lectin pathways of complement

• MBL is a collectin, and is one of a number of serum proteins collectively known as

opsonins. MASPS (Mannose-binding lectin-Associated Serine Proteases) couple
collectins to the complement pathway
• Before ligand binding, MASPs lack protease activity
• Binding of multiple carbohydrate recognition domains of MBL to the microbial cell
surface leads to conformational changes in the molecule, which promotes
proteolytic activation of the MASPs, which then initiate the complement cascade

Defence against Candida infection

• Activation of complement results in phagocytosis by neutrophils and macrophages

• Phagocytosis triggers the production of ROS
• Interactions of cell wall components such as mannoproteins and b-glucan with
phagocyte receptors such as TLR2 and dectin-1 stimulate release of cytokines
including IL-1, IL-6 and TNF
• Production of cytokines by activated neutrophils, together with direct interaction of
neutrophils with dendritic * within inflammatory sites, promotes the maturation of
dendritic cells, such that adaptive immunity becomes activated
• All of these happening at the same time to increase recognition and to deal with the
cells

ROS and phagocytosis by a neutrophil

ROS break up the cell wall
Chromoblastomycosis and Fonsecaea pedrosoi

• non-fatal disease of skin and subcutaneous tissues

• frequently occurs in tropical and sub-tropical regions of America, Asia and Africa
• infection occurs following transcutaneous trauma (mostly farmers)
• chronic disease that can take decades to develop (polymorphic skin lesion)
• difficult to treat (anti-fungals, surgery)

• Chronic fungal infection

• Some of these pathogens are good at not activating TLR and Dectin-1
• Signal through other components that arent partically good at increasing immune
response.
• Chromoblastomycosis caused by fonsecaea.
• Tropical regions of south america, moist, endemic, someone gets a scratch or cut,
and then over many years, the infection spreads across the body.

• Pam3CSK4, synthetic agonist that activates TLR2, activates macrophages when there
is f.pedrosi. Potential treatment.

Parasites - Helminths

• Large, multicellular, organisms

• Generally don’t replicate within the mammalian host - passage through intermediate
hosts or through soil or water required
• Repoduce sexually to create larval stages that mediate transmission
Consist of:

• Nematodes (roundworms)
• Trematodes (flukes)
• Cestodes (flatworms or tapeworms)

• Cause intestinal or systemic infections in humans

• Responsible for massive morbidity especially in developing countries
• Hookworms, Loa Loa, Schistosoma
Schistosomiasis

• Water borne snail is the vector

• Snail is the intermediate that produces the next stage of the life cycle to infect
humans
• Produce eggs
• Schistosoma are the most important parasitic trematode, or fluke, infection of
humans
• Infection occurs in fresh water contaminated with motile, fork-tailed cercariae

Life cycle of schistosomes

• In fresh water, eggs hatch to yield motile

miracidia
• Miracidia infect snails
• In their intermediate snail hosts, the
miracidia undergo maturation and
multiplication to release many infectious
cercariae, thus completing the life cycle

Those then attach to legs, get in blood stream, goal

is to get to liver. Transported through the
circulation to get to the liver, lay eggs, which then
extrecreted through the bladder or intestines, and
those eggs come out and turn into miracidia which
infect snail and go around the life cycle.

Organisms shed their tails and use a combination of

proteases and the muscular action of their oral
sucker to penetrate the host’s skin
Loses tail region, the top bit breaks through skin
and into the blood stream

Cercariae→Schistosomula→Schistosomes

• After penetration, immature schistosomula migrate via the circulation to reach the
liver microcirculation, where they mature.
• Worms do not multiply in the host - adult females reside intimately within a canal in
the ventral body of the male
• Pairs migrate against the portal venous blood flow (GI tract to liver) to release eggs
into the veins draining the bladder and intestines
 • Eggs elicit granulomatous inflammatory response that is believed to assist their
transit into the intestines or bladder, and excretion from the body in the stool or
urine respectively
• Shedding of the egg that causes the damage as it causes the immune response.
Schistosoma Infection Cycle

• Snail produce miracedia, produce cercaie, penetrate skin, get into bloodstream to
become schisosomula, first through lungs and into heart, then into the liver
microcirculation. Worms mature as males and females produce eggs, they move to
the messenteric vessels of bowel or bladder where females lay eggs.
• Some of the eggs get taken back up to the liver and within liver you get damage
which leads to death. (this part that causes damage)
• Majority of the eggs excreted, go back into snails.

Granulomatous responses to schistosome eggs

• Immune responses during early stages of infection are directed against
schistosomula, and demonstrate a TH1 profile
• egg laying, Th1 (activate macrophages) shifted to vigorous Th2 (activates b cells to
produce antibodies), IgE.
• Characterised by tissue eosinophilia, elevated IgE and a TH2 pattern of cytokines, the
cardinal feature of schistosomiasis is the presence of tissue granulomas surrounding
eggs
 • Once get Th2 response, elevelted IgE produced by IgE producing b cells and Th2
pattern of cytokines, B cell activation.
• All of this comes together to surround the eggs as a attempt of the adaptive immune
system to deal with the eggs, That causes problems.
TH1 and TH2 profiles

Th1 – initial t cells activated in the adaptive

immune system to parasites, producing
cytokines which activate macrophages

Th2 – during egg laying, stop macrophages

and activate B cells to produce IgE, to
degranulate eosinophils at the site of the
parasite.

Type 2 granulomatous responses to schistosome eggs

• Granulomas are characterised by an organised circumferential infiltrate of TH2 cells,

eosinophils, macrophages and fibroblasts within a dense collagen-rich matrix
• These are designated type 2 granulomas
• Granulomas are translocated into the intestinal lumen for excretion
• Eggs that are swept into the liver become trapped in the hepatic sinusoids – the
granulomatous response sequesters toxic egg antigens and kills the eggs
• The granulomas slowly dissipate, the collagenous scars coalesce and cirrhosis
develops leading to obstruction of blood flow and the creation of aberrant vascular
by-pass channels called varices
• Death occurs from bleeding varices

• During the whole process of shedding, some of the eggs, rather than being excreted
are taking up to the liver.
• Most come out through the gut or through the urine but some go into liver.
• This causes damage as you get type 2 granuloma.
• Type 2 granuloma is the infiltrate of different cells and antibodies in an attmept to
deal with the eggs. You get ball formed by th2 cells, esoinophils, macrophages and
fibroblasts, where it is this large body of cells that causes damage within the liver.
The granuloma slowly dissipate as the egg is taken apart by the different cells and
antibody. During that, the liver has to produce extra blood vessels around the
 granuloma to deal with the blockage. You then get cirrhosis of the liver. (same as
chronic alcohol exposure).
• You get bleeding varicies which lead to death.

Parasites - Protozoa

• Diverse group of unicellular pathogens that typically cause chronic infections,

indicating a high degree of evolutionary adaptation e.g. malaria
• Antigenic variation is a common theme that underlies the capacity of these
organisms to persist in the host
• The impact of protozoal infections on human health can only be estimated, but it is a
massive global problem

Divided into 2 groups:

1. Those introduced by bites or tissue injury
2. Those introduced by ingestion of contaminated water

Parasites – Protozoa
Trypanosoma

Trypanosoma cruzi

• Trypanosoma cruzi
• Chagus disease.
• Vector -> kissing beetle.
• Vector that moves next stage of the life cycle back into the human.
• Endemic region is south America, where it causes mega colon.

Trypanosoma brucei

• African sleeping sickness.

• Trypansoma Brucei.
• Tsete fly takes a blood meal from a infected person, reinfects a healthy individual,
the parasites migrate to lymph node to the back of the neck. Break the brain blood
barrier, get into the brain.
• Get swelling of the brain and get lethargic.

Trypanosome Variable Antigen Types (VATs)

• In trypanosomes, periods with few parasites (and disease symptoms) are followed
by a large increase in parasite population
• This cycle tends to repeat itself until the host dies or becomes asymptomatic
• Trypanosomes have evolved an amazing mechanism for escaping obliteration by the
host’s defences - antigenic variation, resulting from the successive dominance of
each of a series of variable antigen types (VATs) over time
• Remissions appear to result from generation of protective antibodies that destroy
the homologous trypanosomes
 • Each time the host’s antibodies are almost successful in eliminating infection, the
parasites elude destruction by expressing a new variant-specific surface glycoprotein
(VSG), thus becoming a new VAT, and then rapidly multiplying

• Produce VATs via a thing called VSG. Varient specific surface glycoprotein

• In patients, periods with few parasites, then followed by a large increase in parasite
population.

• Cycle repeats until the host dies or become asymptomatic.

• Antigenic variation, using escape mechanism where the immune system can no
longer recognize them and has to catch up to deal with new variable antigen type.

• Over time you get production of different antigen types, b cell and antibody have to
catch up, parasite number drop, patient becomes asympamatic, parasite mutates,
and then repeat.

Expression of VATs

• Expression of VAT genes occurs in an imprecisely predictable order

• Furthermore, when ingested by a tetse fly, the parasites lose their VSG surface coat
• Expression resumes when the trypanosomes reach the metacyclic state and are then
able to infect the mammalian host
• The VAT phenomenon is best known in T. brucei, but antigen switching is also found
in T. vivax (animal trypanosomiasis)
• The use of surface proteins as counter-defences against host immunity are also
known in several other groups of parasites

• Cant predict it, cant control it.

• In tetse fly, it loses the VSG surface coat, only when it has a blood meal, it coats itself
in VSG surface coat. Switching it on and off in the vector.

Variable Antigen Types

• Within blood stream it is VSG positive.

• When it is taken into the tsete fly as blood meal, it turns of VSG.
• When go back to feed, it then goes back to VSG positive.
• Human has to then go back to deal with it.
VSG activation

• The VSG recognised by the host’s immune

system is released through the flagellar
reservoir and completely covers the parasite as
a surface coat
• Each T. brucei individual possesses ~1000 genes
coding for VSGs (~20% of the genome) - only
one VSG is expressed at any time - the others
are transcriptionally silent
• VSG genes are expressed only when at the ends
of chromosomes, in special telomeric sites
known as VSG expression sites

• Only 1 site of the chromosome where it does it.

• The VSG recognized by the host’s immune

system is released through the flagellar
reservoir and completely covers the parasite as
a surface coat

• As it goes through different switches, the

immune system, produces a type of anti VSG,
drops off, contained, switches VSG, immune
system gets new anti-VSG

The Immune Response to T. brucei

• Initial immune response to infection by T. brucei characterised by early release of
inflammatory cytokines (IFN-g and TNF) associated with a TH1 immune response
• Macrophage activation is a hallmark of infection with trypanosomes
• Numbers and activities increase dramatically in the tissues of trypanosome-infected
hosts
• VSG-specific, B-cell responses associated with temporal immunity to the
trypanosome VATs arising during chronic infection
• T-cell-dependent immune responses to invariant components of the VSG molecule
associated with resistant hosts
• Sum and interaction of these immune responses thought to determine relative
resistance and susceptibility of the host and outcome of infection

• VSG specific B cell response occurs, you get temporal immunity, switch of VSG, loses
the immuntity of B cell.
 • T-cell dependent immune response of the invariant part (the protein part) (not
glycoprotein part), that doesn’t change is what gives the hosts immunity.

Lecture 8 – immune reaction to viral infection

Viruses

• Transient
• Most common recurring infections in humans. Especially this time of the year
• Responsible for the commonest recurring infections of humans
• Although usually transient, they have massive economic consequences
• Viruses that have ‘jumped’ species barriers (zoonotic) generally cause substantially
greater mortality than endemic viruses that have adapted over greater evolutionary
time to their hosts
• HIV from chimpanzees in Africa
• Hantaviruses from rodents in North and South America
• Severe Acute Respiratory Syndrome (SARS) coronavirus from bats in China

Viral Diversity

• Exist as obligate intracellular pathogens - dependent on host proteins for replication

• May contain as few as 3 to 4 to up to several hundred genes expressed from single-
or double-stranded RNA or DNA genomes
• Genomes are enclosed within capsids (coat proteins)
• Cytopathic (lytic) viruses e.g. poliovirus and influenza virus, lyse host cells by
inducing apoptosis or autophagy
• Some cytopathic viruses e.g. herpes viruses can infect cells without producing
infectious virions until immunity wanes or until reactivated by various physiological
signals - known as latent viruses
• Non-cytopathic viruses e.g. hepatitis B replicate without destruction of cells

• Request host cells to replicate and spread, use our machinary in our cells
• Introduction their genes in our cells, take everything in our cells to take advantage.
• Genomes are enclosed within a capsid / coat protein where the immun system
recgonises this
• Cytopathic -> destory the cell when they come out (apoptosis or autophagy)
• Herpes -> infect cells, sit there for many years. Infected much early on, bit when you
get ill or stressed, the virus then wakes up. Latent virus to wait for immunity to wain.
• Non-cypopathic viruses, such as Hep B, replicate without destruction of cell
Breaking and Entering

• Like bacteria, viruses must overcome epithelial barriers of skin and mucosa to
establish infection
• Intestinal viruses e.g. poliovirus enter via antigen-sampling
• M cells
• Respiratory viruses e.g. influenza virus and rhinoviruses establish infection in the
epithelial cells of the airways
• Some viruses e.g. dengue virus and West Nile virus require insect vectors for
transmission
• Bloodborne viruses e.g. HIV and hepatitis B can invade through mucosa or epithelia
following physical trauma

Tissue-specific Receptors and Viral Tropism

• Viruses exploit specific molecules on cells as receptors for invasion

• While some viruses bind widely expressed surface molecules, most viruses invade
using receptors with restricted tissue expression patterns e.g. HIV
• They are said to display TISSUE TROPISM

Tissue tropism is the cells and tissues of a host that support growth of a particular virus or
bacterium. Some bacteria and viruses have a broad tissue tropism and can infect many
types of cells and tissues. Other viruses may infect primarily a single tissue. For example,
rabies virus affects primarily neuronal tissue.

• Use receptors on the outside of cells to infect cells. Some of them use a wide variety
of surface molecules. Most viruses invade using receptors with a restricted tissue
profiles.
• HIV display tissue tropism, uses specific sets of receptors, targting specific receptors
on the outside of specific cells to get into the body and to infect cells
• HIV -> CD4 found on the outside of T cells, CXCR4, CCR5 and CD209 (outside of DC
cells)
• Targeting specific host receptors to get in the cell or to get across membranes into
the body, this is tissue tropism

Immunity

• Major innate immunity is mediated via type 1 interferons*, complement and natural
killer (NK) cells
 • Major adaptive immunity is mediated via antibody and cytotoxic T lymphocytes
(CTLs)
• *Group of proteins that induce host cell enzymes that affect transcription and
translation of viral genes
Adaptive Immune Response to Acute Virus Infection

• Appearance of virus is followed rapidly by a massive expansion of

virus-specific CD8 T cells
• CD4 T cell expansion and contraction also occurs but at a lower magnitude
• Neutralising antibodies are produced relatively early after acute infection with
cytolytic viruses

Timeline
• Massive proliferation of virus,
followed by CD8 t cell
response, aided by CD4
response.
• If exposed before kicks in
very fast, within days, you
start to get death of cells
infected with the virus by kill
cd8 t cells.
• Aided by cd4 t cell specific for
that cd8 t cell, stimualte cd8
killing by cytokines.
• Neutralizing antibody and b
cell production, acute
infection, builds up fast in
line with CD4 and CD8. In a
chronic infectino, neutralizing
antibody take a long time to
build up.

Mechanisms of protective neutralising antibody responses to viruses (compliment and NK

cell mediated ADCC)

• In terms of immunity, complement and antibody.

• Antibody binds to virus, activates complement (classical activation).
• Viral particle gets peppered with compoenents of complement, viral particle picked
up by compleenent receptor on the outside of macrophage, and leads to
phagocytosis of the entire complex.
 • Macrophages have Fc receptors, binds to neutralizing antibody that is opsonizing the
outside of the virus particles.
• Complement activation by classical pathway and the macrophage fc receptor way
• 2 ways

The C1q/r/s complex couples antibody binding to the classical pathway of complement Its
structure is closely analogous to MBL and H-ficolin complexes with MASPs (lectin pathway).

Natural Killer Cell-Mediated ADCC

• Target cell uninfected, produced a b cell response neutralizing antibody response,

that is binding to outside components that are going to bind to receptors on the
outside of the cells. Neutralizing antibody block binding of the virus to receptors it is
using tissue tropism to get in the cell.
• Infected target cell, viruses replicated and coming back out, those same antibodies
can bind to the the Fc receptor on the outside of the NK cell, which will stimulated to
kill the the infected cell. Does it the same way as CD8 t cell, using perforins and
granzymes.
• Antibodies important, block binding of the receptors and recognize the virus coming
up and bind to NK cells to kill the infected cells.

Epidemics and Pandemics

Epidemic: classification of a disease that appears as new cases in a given human population,
during a given period, at a rate that substantially exceeds what is ‘expected’, based on
recent evidence

An epidemic may be restricted to one locale (an outbreak), more general (an epidemic) or
even global (pandemic)

Pandemic: an epidemic that spreads through human populations across a large region e.g. a
continent, or even worldwide

AIDS and HIV

• Acquired immune deficiency syndrome (AIDS) is caused by the human

immunodeficiency virus (HIV)
• HIV infects and destroys CD4+ T cells
• HIV-1
• People get PCP
PCP or pneumocystic pneumonia – lung infection in immunocompromised patients,
treatable but have to be early.

Human Immmunodeficiency Virus-1

HIV-1 originated in southern Cameroon from a simian immunodeficiency virus (SIV cpz), a
retrovirus that infects non-human primates e

Zoonotic movement

SIVcpz introduced into humans through bushmeat activities with subsequent mutation into
HIV – requires one or more high-risk transmission channels for human-to-human spread
Channels absent in Africa prior to 20th Century but growth of large Colonial African cities led
to societal changes such as prostitution (with increased frequency of STDs (genital ulcer
diseases e.g. syphilis)) that allowed sexually transmitted infection

Origin of HIV/AIDS Pandemic – 1920s Kinshasa

Human
Immunodeficiency Virus

GP120, docking
glycoprotein

GP41, transmembrane
glycoprotein

Docking with receptors,

allowing movemement of
viral particle into the cell.

HIV Epithelial Entry

• HIV-1 can cross mucosal surfaces through M cells - prevalent in tonsils and rectal
epithelia
• It can also gain access via epithelia damaged by ulcerative infections, or by trauma or
injection (i.v. drug use or blood transfusion)
• Vaginal epithelia lack M cells - HIV gains access via interdigitating processes on
Langerhans cells (-> cells sticking finger like projections out and sampling for antigen)
 Epithelial cells in the small intestine express
CCR5, a chemokine receptor HIV-1 binds to CCR5 (once tethered by
galactosylceramide) via the viral envelope glycoprotein gp120 and the virus is
transcytosed

HIV Transport to Lymph Nodes

• Once across mucosal epithelial barriers, HIV-1 encounters sub-epithelial dendritic

cells
• C-type lectin receptors (CLRs) on dendritic cells bind high-mannose N-linked
oligosaccharides on viral envelope glycoprotein gp120
• Interaction with CLRs initiates dendritic cell migration to regional lymph nodes
• Once in lymph nodes, intact virus, internalised with CLRs, is re-exposed at the
surface and displayed to T cells
• Regardless of the route of infection, the predominant site of virus replication early in
disease is in the small intestine - this reflects the large number of activated T cells
that express CCR5 in that organ

• Entry of HIV into cells mediated by viral envelope glycoprotein gp120

• Binds to CD4 and chemokine receptors (CCR5 or CXCR4) on host surfaces
• Binding with receptor leads to a conformational change in gp120 that exposes the
transmembrane component of the envelope protein gp41
• gp41 mediates fusion of the virus envelope with the host cell membrane

HIV-1 Infection Cycle

CD4 co-receptor on the outside of the CD4 t cell.
GP120 binds to it, get conformatinal movement to allow gp41 to interact with chemokine
receptor CCR5 and once that happens, the whole thing is taken into the cell.

HIV infection mechanism

• HIV infects cells via 2 cell surface molecules, CD4 (primary receptor) and chemokines
CCR5 and CXCR4, act as co-receptors for the viral infection for macrophages and t
cells respectively.
• Hiv binds to CD4 with GP120 (envlope glycoprotein), interaction with virus with CD4
and the co-receptor allow virus uncoding and the entry of the nucliocapsin
containing the viral genome into the cell.
• The viral reverse transcriptase which is a part of the viral particle, copies the RNA
genome of the HIV into dsDNA, the viral integrates then mediates the integration of
the viral DNA into the chromosomal DNA of the host cell.
• In this stage, the virus is latent, it can persist in the cell in an inactive state.
• Reactiation of the virus occurs when the host cell is activated and viral transcription
is activated. This results is accumulation of viral proteins, as well as genome linked
RNA transcript of the virus.
• Viral proteins assemble at the cell membrane with copies of the RNA genome, and
bud off to create a new viral particle.
• Maturation of this new virus particle continues after it is budded off from the host
cell, to create a new infectious virion with its characteristic nucleocapsin
morphology.

Immunological Consequences of HIV Infection

• Dendritic cells translocate HIV from mucosa within 30 minutes of infection

• A wave of viral proliferation in lymph nodes peaks 4-7 days after infection
• Viremia peaks at 14 days and all lymphoid tissues are infected by 3 weeks
• HIV is quiescent in resting CD4 T cells, but on T cell activation, virus production is
also activated and the host cell dies
• >99% of virus is produced by newly infected CD4 T cells
• ~107-108 CD4 T cells are producing virus at any given time
• Loss of helper activities (mainly through cytokine stimulation) means that
maintenance of cytotoxic CD8 T cells is lost as is the antibody response of B cells

• Viremia (production of virus)

• Quiescent, rest in CD4 t cell and doesn’t do anything. Only when particular t cell
activated, it goes through clonal expansion, you get virus being actiavted and the the
host cells die.
• Loss of helper t cell actives critical, lose very cell of the adaptive immune system that
monitors everything and helps in destruction.
• Means maintanance of cytotoxic cd8 t cells and antibody response is lost.
Progression of Untreated HIV Infection

• Infection typically results in flu-like illness from days to weeks after exposure -
associated with a drop of CD4 T cells in the blood
• The CD4-dependent antiviral cytotoxic CD8 T cell response develops and is
responsible for a prolonged period of stable viremia
• This is known as the VIRAL SET POINT
• Patients are normally asymptomatic until the CD4 T cell counts decline further -
infection of developing lymphocytes in the bone marrow and thymus results in
failure to replace lost T cells
• Opportunistic infections begin when CD4 T cell counts reach levels <200/ml
• Death then typically ensues in about 2 years

First few weeks -> big peak in viremia, drops off, stable population kept in check by CD8 t
cells and neutralizing antibody. CD4 t cell population rumbling.
CD4 t cells crash in later stages and then you are in trouble, virus takes off, lose CD8 t cell
response, lose antibody response, get secondary nasty infections.
AIDS and Microbial Infection diseases
Herpes, candidiasis, cryptoccocal meningitis, TB

Cryptoccocal meningititis

• Hard to treat
• Fungal
• Aids defining disease caused by cryptococcus neoformans and gattii.
• Can sometimes affect healthy people, but very prevelant in patients with AIDs.
• Opportunisitc infection.

Influenza

Influenza Viruses

• There are 3 classes of influenza viruses - A, B and C

• Influenza A and B are major causes of human disease
• Only influenza A is responsible for pandemic outbreaks
• Influenza A viruses are avian viruses that adapt to humans
• The virus is spread by aerosol droplets and establishes cytolytic infections in the
epithelial cells of the upper and lower respiratory tract
Influenza A viruses

• Influenza A is a spherical
or rod-shaped enveloped
virus covered with 2
spike-like glycoproteins:
• Trimeric hemagglutinin
(HA)
• Tetrameric
neuraminidase (NA)
• HA mediates binding to
cell surfaces and
internalization
• NA cleaves sialic acid and
promotes viral release
from cells

Hemaggultinin
Neuraminidinase (allows it come back out) Uses enzyme to break link of the cell then that
virus can go on and infect other cells

HA, NA, Binding and Budding

• Infleunza A virus, HA, surface receptor, binds to sialic acid (or neuraminic acid),
which is a carbohydrate bound within the receptors of the epithelial cells. Binds to
sialic acid, via HA receptor, once that happens, it endocytosed, uncoating, RNA
replication, production of viral proteins, put back into coat protein, it buds off and
finds another cell.

Influenza Genotypes
• Influenza genotypes are diverse in birds (migratory waterfowl) and express a variety
of HA (H1 to H15) and NA (N1 to N9) subtypes
• Avian viruses prefer receptors, primarily on intestinal cells, that have the a-2,3 sialic
acid linkage to galactose
• Human viruses prefer the a-2,6 linkage expressed on respiratory epithelia
• Pigs express both types of linkage on respiratory epithelial cells
• Influenza viruses enter the human population in regions where humans, pigs and
waterfowl, as wells as domestic chickens, are in close proximity
• Pigs can be infected with both human and avian influenza, and exchange of
segments between viral genomes in the pig can give rise to variants expressing novel
surface proteins together with human-adapted virulence determinants

Antigenic SHIFT and Antigenic DRIFT

 • Epidemics occur when point mutations (red dots) accumulate in surface HA (ovals) or
NA (diamonds)
• This leads to ANTIGENIC DRIFT so that, in many previously immune individuals, HA
and NA are no longer recognised by neutralizing antibodies produced in response to
earlier infections
• Pandemics occur when avian influenza viruses and human influenza viruses infect
pigs and re-assortment of the two genomes leads to the expression of avian HA or
NA in a virus otherwise adapted to infect humans (ANTIGENIC SHIFT)

Antigenic DRIFT

• Infleunza have receptors which can bind to host cell surfaces

• Neutralizing antibodies stop influenza to bind and infect cell.
• Some virus have mutation that later receptor that stop the neutralizing antibody
from stopping it from binding and infecting the host cell.
• The Pattern of antigens expressed by the virus can change over time, called antigenic
drift, contributes to susceptible infleunza infections every year

Antigenic SHIFT
• Neutralizing antibody so that virus cannot bind to cell.
• In some causes, viruses arise that can escape the effects of a neutralizing antibody.
• Happens when 2 different strain of infleunza able to infect the same host cell.
• The combined viruses can contain segmants of genome from either of the 2 original
viruses.
• Some viruses will acquire a segment of a genome from the other strain, encoding the
receptor from host cell surfaces.
• Neutalizing antibodies that can originally bind to the original virus, will be unable to
recognize the receptor from the 2nd strain, and will be unable to prevent the virus
from binding and infecting host cells
• This process where large changes of antigenicities is known as antigenic shift
• Much of the immunity against the original virus is ineffective.
• Such antigenic shift mutations are often to do with large scale virus pandemics.

Immune Response to Influenza Virus Infection

• Antibody produced against HA and NA no longer able to block binding to the
receptor in the epithelial of the lungs (NA)
• Antibody produced against NA can no longer tether the viruses that come out and
prevent their movement against new cells for infection.
Influenza Pandemics

• Human infections are generally but not exclusively limited to viruses that express H1,
H2 or H3 and N1, N2 and possibly N8 subtypes
• Three global influenza pandemics occurred in the 20th century

1918-1920 ‘Spanish’ flu pandemic (caused by H1N1)

1957-1958 ‘Asian’ flu pandemic (caused by H2N2)
1968-1970 ‘Hong Kong’ flu pandemic (caused by H3N2)

A 21st century global influenza pandemic analogous to the 1918-19 outbreak is thought to
be inevitable

‘Spanish’ Flu
• The 1918 flu pandemic, was cause by an unusually severe and deadly Influenza A
virus strain of H1N1
• Many of its victims were healthy young adults (>50% in the 20 to 40 yr-old age
group), in contrast to most influenza outbreaks which predominantly affect juvenile,
elderly, or otherwise weakened patients
• This was due to extremely high infection rates and the extreme severity of
symptoms, suspected to be caused by hypocytokinemia,

Hypercytokinemia

• A ‘cytokine storm’ (hypercytokinemia) is the systemic expression of a healthy and

vigorous immune system
• Caused by release of >150 inflammatory mediators (pro-inflammatory cytokines,
anti-inflammatory cytokines, free radicals, coagulation factors etc.)
• Precise mechanisms for cytokine storms are not known, but may be caused by an
exaggerated response when the immune system encounters a new and highly
pathogenic invader
• They have the potential to do significant damage to body tissues and organs e.g. if in
the lungs, large concentrations of macrophages accumulate restricting the airways
• Cytokine storms occur in a number of infectious and non-infectious diseases e.g.
avian influenza, adult respiratory distress syndrome (ARDS), sepsis and Ebola

Cytokine storm
- Expression of healthy immune system
- Massive cytokine production, huge damage to lung tissue.
- People died within a night time of getting the infection
- Drowned in bloody mucus
- Massive production of alveolar macrophages, cytokines that destory lung epithelia,
you troth up this bloody mucus

Why is a 21st Century Flu Pandemic Inevitable?

 • Highly Pathogenic Avian Influenza (HPAI) viruses, which express H5, H7 or H9, have
become established in poultry throughout China and Southeast Asia
• HPAI H5N1 viruses were transmitted to individuals exposed to poultry during
outbreaks in Hong Kong in 1997
• These have spread, primarily through migratory birds, to infect poultry and humans
in the Middle East and northern Africa
• Human transmission of H5N1 is infrequent, but mortality approaches 60%
• Pigs in China have been infected with avian H5N1 strain and the human H3N2 virus
strain (responsible for the 1968-70 HK pandemic)
• This may facilitate the re-assortment of viral genome segments to produce a more
transmissible virus

Only way to prevent it is vaccination.

Ebola
Wild bats that get into chimpanezees
Probably bushmeat
Carry Ebola virus
Associated with wild bat population in africa

Simple horrible looking thing

• 7 genes in its genome

• Everything that is required to replicate in the human host is there.
• 1 important gene incodes this thing called ebola virus glycoprotine (GP) has a
membrane bound found, and a soluble secreted form.
• Membrane bound form allows it to target receptors on the outside of monocytes
and macrophages, which leads to massive cytokine disregulation. Inflammatory
cytokines produced leads to break down of cells and hemerrhaging within the body
• Soluble dimeric form is secreted after it has infected the cell and prevents neutrophil
activation.
• Taking out predominatly the cells of the innate system. Nasty, really fast, have to
contain really fast.
• Simple organism that causes massive damage to humans.
Lecture 10 11 and 12 Vaccines

Diptheria, Measles, rubella,

Andrew Wakefield doctor published reported in 1998

Claimed mmr vaccine caused autism
Distrust of MMR vaccine
People stopped vaccining MMR
So there was increase in MMR

• No controls included.
• Autism would occur in 25 non-immunised children/month based on probability
• No experimental evidence for the hypothesis that MMR peptides were translocated
to the brain

H1N1 Flu vaccine and narcolepsy

Risk-benefit analysis for vaccines

• risks – side effects (lots of people develop some sort of local reaction, reddening,
swelling after a day), allergic reactions,
• compare to other probability
• Side effects being infected compared to side effects of measles vaccine
Main types of vaccine

Sub-unit vaccines

Isolated components of a microorganism

e.g. an individual protein
e.g. an individual polysaccharide

• Fragments of microorganisms.
• In general they are molecules on the surface of the microorganism
• Might be protein to do with invasion, adhesion, might be polysacchrides involved in
enabling bacterium to prevent phagocytosis, might be components of type 3
secretion system
• Probably only 1-2 will work well as components of the vaccine.
• Challenge is to find out which bit you need to include

Immunity induced by sub-unit vaccines is almost always antibody-mediated

Single sub-unit vaccines
• In some cases an immune response against a single antigen elicits protection
• Diphtheria is caused by the bacterium Corynebacterium diphtheria
• The bacteria produce a potent toxin which blocks protein synthesis and causes heart
failure
• Neutralise toxin = stop disease
• Vaccine prepared from diphtheria toxin, treated with formaldehyde
• Tetnus is also formaldehyde toxoid
Formaldehyde detoxification

• Formaldehyde cross links amino groups in proteins with other nearby nitrogen
atoms in protein through a -CH2- linkage
• Both intramolecular cross-linking and cross-linking between adjacent protein
molecules
• Forms methelyne linkage (CH2) linkage between adjacent immunogroups in
proteins.
• Forms linkage which would not noramlly occur. Linkage within the protein molecule
or it could be linkage between adjacent protein molecule.
• We lock structure of the protein by cross linking it and link lots of molecules together
so they become a large complex. Physically change structure of protein so it cannot
exert its toxic action, but preserved the structure of the protein sufficiently, so
immune system will see it and recognize it, and produce antibodies against they
cross linked protein that will react to that toxin
• LYSINE AND GLUTAMINE
 • The toxicity of the protein is abolished
• The immunogenicity of the protein is preserved
• Individual diptheria toxin, cross link by formaldehyde, end up with complex,
physically prevented from exerting toxic effects.
• Take purified toxin, treat it with formahyde so it cross links, and denature it, and it
can be used as an immunogen.

• Problems;
– Must balance cross-linking with retention of antigenic structure
– Batch to batch variation
– Reversion
– Requires pure toxin as the starting point
• Need to grow pathogen
• Need to purify and handle the toxin

A modern approach to subunit vaccines

• Modify the toxin using genetic engineering so that it is non-toxic but immunogenicity
is retained
• Genetic toxoids

Diphtheria toxin CRM197 genetic toxoid

• Mutation in the catalytic A-subunit blocks activity

• glycine to glutamic acid substitution at residue 52
• Example of genetic toxoid against diphtheria toxin.
• By introducing mutation into the catalytic subunit, it abolishes the catalytic activity
• By introducing glycine to glutamic acid substitution at residue 52, it produces a
protein that looks identical to toxin, but it non toxic.
• Cant revert
• Not hazardous
• No batch to batch difference
• Very reproducible

Necrotic enteritis
 • Necrotic enteritis is a problem is poultry.
• Problem that is servere in the past decade.
• In the past antibodies are thrown in animal feedstuff to stop animal infection.
• Consequence of antibiotic resistance, there are various bits of legislation that
prevent antibiotic in animal feedstuff.
• Necrotic enteritis appears from this. It punches holes in the gut. Reason it is
perforated, a toxin is produced in the gut that physically breaks down the gut wall,
damages the gut, allows leakage of material out of the gut. Fatal disease.
• Toxin breaks down gut

NetB toxin causes Avian Necrotic Enteritis

NetB forms pore, leads to inability of the cell to regulate osmotic balance, which kills cell.

• Seven NetB monomers assemble into a ring-like structure

• Inserts into the target cell membrane, forming a pore
• Disease is NetB toxin. Consists of is 7 individual monomers of the toxin which
assemble into a mushroom shaped structure. Has hole down the middle
• This complex can insert into cell membrane, in cells lining the gut. The stem region
inserting across the membrane, now there is a pore which connects inside and
outside of the cell. Allowing ions moving in and out of a cell in a non-controled way.
• If we can neutralise toxin, we can protect against the disease.

• Inserts into the target cell membrane, forming a pore

• Inability of the cell to regulate osmotic balance kills cell
• Neutralise toxin = stop disease

NetB toxin genetic toxoid

• Identify amino acids which are critical for toxicity (e.g. W262)
• Produce W262A with abolished toxicity
• Test as a vaccine
• W262, mutate it and abolish toxicity, and so the toxin cannot bind to the cell
membrane.
• If we change that residue we stop binding.
• Potentially use as a vaccine.

Advantages of genetic toxoids

• Produce in a harmless bacterium (e.g. E. coli)
• High yields – ease of purification
• Molecular structure more similar to the active toxin
• Reproducible properties
 • No reversion
• Cheaper

Multiple sub-unit vaccines

Plague & Yersinia pestis

• Caused by the bacterium Yersinia pestis
• The Justinian plague
– AD542 - AD570
• The black Death
– 14th - 16th centuries
• The third pandemic
– mid 19th century - ?

A sub-unit vaccine against plague

• Identify the key protective components

– F1-antigen - capsule
– V-antigen – part of the type III systems
• Produce F1 and V antigens using genetic engineering

• Identify proteins on the surface of proteins.

• Indentified F1 which formed the capsule around bacterium to prevent from being
phagocytosed
• V-antigen is the part of the type 3 secretion system (tip of type 3 system)
• Type 3 secretion system – molecular syringe structured in the bacterial cell wall,
poke out on surface of the cell that injects toxic proteins in human cell

How do we find the protective protein sub-units ?

• Decades of research on understanding how the pathogen causes disease

• Trial and error !
• Research on how they cause disease, which proteins are on surface, what proteins
do they use, what are their architecture. Need to know these before testing on
vaccine.
• Bit trial and error. Put a bit of antigen on mouse and see if you can protect mouse
from disease. There is a better way, through reverse vaccinology.

Reverse vaccinology

Reverse vaccinology - bacterial vaccines

• Take genome sequene of a microorganism (bacteria or virus), predict from that

genome sequence, which of that protein will be effect to be included in the vaccine
 • Important components that go in the vaccine will be on the surface.
• Predict which on the proteins encoded by the genome will end up on the surface.
Straight forward, as all these proteins which have signatures which will direct their
export on to the cell surface.

pSORT predicts cellular location

• Use pSORT looks at every open reading frame in the genome, and predicts the
structure of the protein, and then asks does it have the right architecture or features
to be exported onto the surface.
• Eg, does it have a signal sequence that directs export, does it have any other motifs,
does it have right amino acid compositions that surface proteins have, build list of
proteins from genome seuqence likely on surface

Neisseria meningitidis B
• Neisseria meningitidis causes globally 1.2 million invasive disease cases and 135,000
deaths per year
• Serogroup B strains are the main cause of disease in Europe, New Zealand, Australia,
Argentina, Canada, Japan
• Traditional vaccinology had failed to identify a vaccine

MenB reverse vaccinology

• Genome sequencing, Men B, sequenced many strains, predicted which proteins
located on surface on bacteria, predicted 570 proteins that might be located on
surface, they produced each of those proteins using recombinant dna technology,
some of the proteins they couldn’t produce for various reasons.
 • They managed to produce 350. They then immunised mice with each of the protein,
they took serum of mice to check if protein was located on surface or if it had
bactericidal activity.
• Mixed serum with bacteria in present of complement to check if antibody could
activate complement mediated killing. Came up with 28 proteins which could be
produced that they could produce, immunogenic in mice, and when tested towards
bacteria, they showed ability to kill bacteria in serum.

Meningitis B vaccine
• H binding protein (fHbp), fused with NA2091 protein, binds human factor H, a
negative regulator of the alternative pathway of complement activation.
• NadA, major adhesion protein involved in colonization, invasion, and induction of
pro-inflammatory cytokines.
• NHBA, heparin-binding protein that increases resistance against the bactericidal
activity of human serum
• PorA 1.4 Porin, A major outer membrane protein and the target for bactericidal
antibodies
• 4 proteins managed to protect men B
• 4CMenB, Bexsero

Vaccines II

Sub-unit vaccines
Polysachariddes

Polysaccharide that are on the surface of microorgniasms (of bacteria)

Compoenents of repeat individual sugars

Combination of different sugars that give rise to different properties of polysaccharide

Many different combinations to give rise to different polymsaccharide.

Polysaccharides in bacteria are located in two surface structures

 • In gram positive, found in polysachharide capsules that sit on surface and cloak
bacteria
• In gram negative, they have capsules in surface also, but they also have LPS, consist
of Lipid A anchor on a polysachharide chain extending out to the surface.
• Surface of these bacteria are covered in polysaccharides
• Different species have differerent polysaccharides

Sugar-coated bacteria
• The different combinations of sugars give rise to immunologically distinct
polysaccharides
• Little or no immunological cross reactivity between different polysaccharides
• Different combinations of sugars linked together that give different immunological
propertities of these polysacharride.
• When you generate antibody against specific polysaccahride, it is very specific for
that conbination of sugars linked together. It doesn’t recognize other
polysaccharides.
• Potentially polysaccharide vaccines are powerful as they are very specific.

Polysaccharide vaccines
• Streptococcus pneumoniae - pneumonia
• Haemophilus influenzae - pneumonia
• Neisseria meningitidis - meningitis
• Salmonella typhi – typhoid fever
• Burkholderia pseudomallei - melioidosis

Streptococcal Polysaccharides and vaccines

• Major cause worldwide of community acquired pneumonia, bacterial meningitis,

bacteraemia and otitis media
• Antibody against the capsular polysaccharide is protective
• In addition to causing local infection, it can cause much more servere systemic
infections such as pneumonia, menigitis, infections can be very difficult to treat, as
when they are diagnosed, they are well developed and well advanced.
• Vaccines prevent disease from being captured in the first place.
• Image – around the cell is the green sugar capsule (polysaccharides)
• Antibodies against the capsular polysaccharides are protective, where they opsonize
them (coat them, make them accessible to phagocytes to ingest them), they also
complement fix and agglutinate the cells.
At least 90 serotypes based on different capsule structures

• Big problems when it comes to developing vaccines against strep Pneumoniae

• Big problem is different strain have different polysachharides on their surface.
• Around 90 different seratypes of strep pneumoniae, each serotype has a different
configuration of polysaccharides on their surface.
• A way in which different strains of bacterium can infect somebody which has
preexisting antibody against a different strain.

Disease caused by S. pneumoniae serotypes

• Technical impossibility to do all 90 serotypes.

• One approach is to include polysachharides from serotypes which are most likely to
cause disease
• Look at different serotypes and frequency they cause diseases.
• 19A especially like to cause disease in humans.

Vaccine with originally only 7 serotypes, now 23 serotypes.

Potential way to solve. Focus on serotypes most likely to cause disease.

Problem 2
• The vaccine causes disease mainly in the very young and the elderly Polysaccharides
are poorly immunogenic in the young and the elderly
• Problem in very young and eldery. Do not respond well to polysaccharides.
• Immune system of the very young cannot see polysacchardies very effectively, not
mature enough to recognise the antigens and produce antibodies against the
polysaccharides\
• IN elderly, the immune system is less efficient and effective, not as good as
recognizing the polysaccharides.
• Works pretty well in our age. Group we want to protect against, works least well.

Polysaccharides are T-cell independent antigens

• Pathways of how proteins are presented to the immune system

• Polysaccharides in these pathways. Not in the antigen presenting pathways proteins
are presented in.
• What it means is you don’t get some of the activities of CD4 cells which are critical
for immune activity.
• No cytokines from CD4 t cell to produce antibodies

When you immunize polysaccharide antigen, you don’t get accessory help, so the antibody
response that develops looks like immature antibody response, predominted by IgM
subclass, very few IgG. Dominated by IgM. Low quality immature response. Good enough to
protect people with functioning and good immune response, but not good enough to
protect young and elderly.

• No involvement of antigen presenting cells, CD4+ or CD8+ T cells

• Polysaccharides interact directly with B-cell
• Clonal proliferation of B-cells
• Production of IgM antibody
• Poor antibody responses in children & the elderly
• no memory

Link polysaccharide to a protein

• Diphtheria toxin CRM197 genetic toxoid

• glycine to glutamic acid substitution at residue 52 abolishes toxicity
• If you link polysaccharides to a Protein, you can modify and enhance their
immunogenicity.
• Physical and chemically link polysaccharide interested in to a protein carrier.
• Converts the polysaccharide to look a little bit more like a protein antigen. One of
the carriers that are in widespread use is this protein, genetically modified form of
diptheria toxin.
• Form which has mutation in the active site region, glycine to glutamic acid region,
where it abolishes the toxicity of the diptheria toxin, Retains all immunogenicity.
Good protein for linking to polysaccharides.

• Take our strepoccocal polysaccharide and chemically couplet to our diptheria toxoid,
to generate this molecule where the polysaccharide chain is physcially connected to
the protein, and this so called conjugate vaccines behave a little bit more like
proteins.

• Switch response to the polysaccharide.

Mechanism of T-cell activation by glycoconjugate vaccines: a new working model

• Glycoconjugate is internalized into an endosome of the B cell.

• processed into
• glycanP saccharides
• peptides
• glycanP-peptides
• MHCII presentation of glycanP-peptide by MHCII to the αβ receptor of CD4+ T cells
(αβTCR).
• Activation of the T cell by the carbohydrate/MHCII results in T-cell production of
cytokines which mature the B cell to become a memory B cell
Polysaccharide conjugates work in children under 2 years old
Incidenced decreased in under 2 years old.

Prevnar conjugate vaccines

• Vaccines tend to used nowadays against strepoccocal vaccines are conjugate

vaccines. Still have to take each polysaccharide to each serotype, and link each one
to a protein carrier, and the conjugates are injected.
• Diptheria toxoid -> protein carrier
• Good to protect against disease
• Major Value in 3rd world countries funded by charities.

Vaccines that induce CD8+ T-cell responses

Diseases where CD8+ T-cells play a role in protection

• Diseases where the microbe grows within host cells.

• tuberculosis
• salmonellosis
• melioidosis
• viral infections - yellow fever – influenza – measles

• Need to kill infected cells through CD8 t cells.

• Cancer vaccines
• Any vaccine where killing of the host cell is required

It is difficult to devise vaccines which induce CD8+ T-cell responses

One approach is to use live attenuated microbes

• Living microbes which are disabled so that they don’t cause disease
• But to work the microbe must be able to survive long enough to establish an
immune response
• Balance between over- and under-attenuation

• How do we evoke CD8 t cell response

• Cant use is same sub-unit vaccines
• Those vaccines go into those APC via the phagocytic pathway, processed by MHC 2
pathway, complex with MHC class 2, activate CD4. This don’t work for CD8.
• Need to get the antigen into the cytosol instead of the endosome and processed in
the MHC 2 pathway.
 • Use microorangism, capable of replicating in the cytosol and use that to vaccine
against the disease. Take a strain of TB that is unable to cause disease but can get
into host cell into the cytosol and replicate, the antigens from the vaccine strain can
become processed by the MHC1 pathway and activate CD8 t cells.
• Problem in using those strains, as we need to balance the strain to grow in the
cytosol and present antigens, with the inability to cause disease. Trade off. Depends
on the health of the immune system.
• Live attenuated vaccines can work very well in healthy people, but risky in people
who are immunocromprimised as those strains can maybe cause vaccines.

CD8+ responses are induced by live attenuated microbes that can invade host cells
• Polio, TB, mumps, yellow fever
• Many live attenuated vaccines in use.
• Microorganism which have been disabled and cannot cause disease in healthy
people, but they can grow and replicate in host cells. Can present protein antigens to
the immune system and stimulate immune response.
• Because they grow in the cytosol, it can activate CD8 t cell responses.
• May not be safe in everybody.

One approach is to use naked DNA vaccines

• Identify and produce gene coding for vaccine component (protein)
• DNA is taken up by the cells in the body (often muscle cells)
• These cells produce the protein
• An immune response develops to this protein
• You vaccinate someone with a piece of DNA which codes for the protein, rather than
with the protein itself. That piece of DNA gets taken up by cells, gets into the cytosol
of those cells, and within the cytosol, that DNA directs the production of the protein.
• Protein produced in the cytosol, and presented to the MHC1 pathway to elicit CD8
response.

• How it works
• Injected, taken up by host cells, into cytoplasm, the DNA is transcribed and
translated so protein is produced in host cell, and the protein is in the right place to
stimulate MHC class 1 to elicit CD8 t cell responses.

Naked DNA vaccines

• In theory combine the advantages of live and sub-unit vaccines
• Work extremely well in mice
• Very disappointing trials in humans
• Problems, DNA might be long lived, difficult to control the amount of protein the
immune system gets exposed to, and also the duration of that exposure. Depends on
how long DNA survives in the cell.
 • Bigger problem, DNA vaccines work well in every species except humans. Significant
limitation in exploiting DNA vaccines in humans.

• West Nile Fever vaccine licensed for use in horses

– Vaccine encodes coat proteins
• infectious hematopoietic necrosis vaccine licensed for use in in fish
– Vaccine encodes surface glycoprotein

Cancer vaccines
• Need to prevent or eliminate cancer cells
• Typically requiring NK and/or CD8+ T-cells
– antigens from tumour cells
– whole cancer cells
– dendritic cells
– Naked DNA vaccines

• Rely on priming the immune system to see the tumour cells and then eliminate
tumour cells. Eliminate them by producing NK and or CD8+ t cell responses.
• Recognize the tumor markers on the cells and elimate them.
• Various ways to do them, immunize with antigens (problem is that they have to get
that in MHC class 1 pathway, get great antibody response, but no CD8 repsonse),
Naked DNA vaccines (not good in humans),
• Try to use dendritic cell vaccines

Expose antigen presenting cells (dendritic cells) to the vaccine outside of the body

• Take dendritic cells from patient, remove them from patient, in the lab, you expose
the DC cells to the antigens you want to prime the response against.
• Use technology to move antigens across cell membrane, electroporation technique
(pulse cells with electric fields, open pores in the cell membrane and those antigens
get into the cell)
• Get the antigens into the cytosol in the APC, so they can go on and elicit a CD8 t cell
response.
• Complex, have to harvest cells from patient, purifiy APC, make sure they are
differentiated into mature DC cells, antigen you want, you have to pulse antigen into
the DC cell, infuse the cells back into the patient, and then the cell will present the
 antigen to the right T cells NK cell, CD8+ t cells to target the tumor cell in vivo.
Hugely expensive, but promising.

Dendritic cell cancer vaccines

• 4 of 14 stage IV melanoma patients showed progression free survival

• Heptocellular carcinoma– 2/12 complete clinical responses, 5/12 stabilisation
• Item right involves selecting APCs specific for tumour antigens, expanding and re-
infusing

Clinical trials.
Very encouraging responses for patients that were beyond recovery.

Vaccine adjuvants

Why are adjuvants needed?

Immune response against time
If you give a protein antigen on its own, or in combination with adjuvent
Intensity and duration is different
Critical to achieve protective immunity

• They increase the magnitude of the immune response

• They increase the duration of protective immunity
• They may allow the type of immune response to be modified
• They may allow lower doses of vaccine to be used
 • Gives you hgiher level of antibody, better t cell response
• Longer, more years instead of few days
• Might allow you to skew the response to one direction (cd4 or CD8)
• Use smaller doses in the first place
• Make vaccines work much better, can use them in combination with proteins,
polysaccharides, conjugate vaccines.

Types of adjuvant

• Depot effects
• Delivery vehicles
• Immune stimulators (modifiers)
• Often adjuvants have combinations of these properties

Depot effects
• The antigen is sequestered at the site of injection, and become released over time
• Exposes the immune system to the antigen over a longer period
• Ensures the protein that is delivered through injection, becomes sequested at the
site of injection so it doesn’t leach away, and it is slowly released through out the
next few days or even weeks.
• Drip feeding antigen to immune system

2 type of depot effect adjuvants

-physically entrap subunit in some kind of shell that breaks down slowly over a period of
time which releases the protein or the polysaccharide from within
-Formulate antigen so it is embedded in a matrix, and that matrix allows the anitgen to
become physically absorbed to it, and subsequently desorbed over a period of time. Slow
release pattern.

Examples of depot effect adjuvants

Aluminium salts – form a gel like matrix, allow the antigen to be absorbed to it, and then
slow released and desorbed, drip fed to immune system
Most widely used adjuvant

Liposomes -> Encapsulate the antigen and over a period of time, the membrane of the
liposome breaks down and the antigen is slowly released
Delivery vehicles
• The antigen is targeted to immune cells e.g. antigen presenting cells
• Delivery vehicles
• Rather than actually slow releasing from site of injection, delivery vehicles try to get
the vaccine antigen into the right cell of the immune system
• Target the vaccine antigen to an APC, so you can get the protein and polysaccharide
into the right cell so it can be effienctly delivered to other cells of the immune
system

Bacterial proteins are processed by antigen presenting cells

Cytosol -> microorgnaism that can replicate in the cytosol -> CD8
Endocytosis -> remain within phagosome, -> CD4
Delivery system are aimed at trying to deliver protein or polysaccharide so you can directly
get it in the APC, and encorportate it in one of these pathways

Particle delivery systems

• Vaccine attached to particles can be taken up by antigen presenting cells (APCs)
• Enter the Class II pathway
• Use virus like particles
• Viral capsid, without any nucleic acid inside, instead you have vaccine antigens
instead.
• Use virus into the APC. Class 2
• Use Liposomes
• Also serve as delivery vehicles, this synthetic cell membrane can fuse with the host
cell membrane and delivery the contents into the host cell, maybe directly into the
cytoplasm (CD8 t cell responses)
• ISCOM
• Polymeric nanoparticle
• Aim is to get the particle into the APC
• Get it into the right pathway in the APC

Liposome delivery
• Liposomes can deliver protein antigens into the cell
• Could enter the MHC I pathway
• Liposome delivery, synthetic bylayer around the surface, and thd antigens instead.
• Liposome fuse with target cell membrane, the vaccine antigen delivers directly into
the cytosol. MHC-1

Immune stimulators (modifiers)

• The adjuvant activates elements of the (innate) immune system to modify the
response
 • Finally, Immune stimulators or modifiers of the immune response
• -provoke immune system into producing the right kind of cytokines and chemoklnes
for an optimal immune response
• Cytokines and chemokines that activate B cells, T cells, APCs,
• Stoke up immune response, so there is more potent antibody or t cell response of
interest

The innate system recognises molecules that are common to microbial pathogens but
absent from the host
• Pathogen-Associated Molecular Patterns (PAMPs)
• The innate system is pre-coded to recognise a limited repertoire of molecules
• This enables it to respond quickly, but only to provide a low level response

• Adjuvants target innate immune system

• These adjuvants switch on innate system, provoke the production of cytokines and
chemokines which allow a more productive adaptive immune response
• To provoke the immune system, we need molecules that are recognized by that
innate system
• Innate system very good at recognizing molecules that are signatures that are
present on the surface of the microoragnism.

Examples of molecules recognised by the innate system

• Peptidoglycan –bcateria
• ssRNA – Virus
• Ds RNA – Virus
• Flagellin – bacteria
• Heat shock proteins – Bacteria
• LPS – Gram negative bacteria

TLR signalling and inflammatory responses

• The adjuvants work by stimulating TLR signalling pathway.
• Structured so they have Horseshoe type domains on the surface of APC, these are
the bits that recognize the foreign molecule.
• Have domains on the inside of the cell that associated with signalling, and
dimerization of these domains are required for the signalling event.
• Need a TLR dimer for signalling. Signalling can occur via MYD88 pathway or the TRIF
pathway.
• These pathways are stimualted seperatly, but there are a lot of cross talk between
the pathways. Once they terminate in, is the production of cytokines and
chemokines, and costimulatory molecules, interferons, and these are molecules that
switch the adaptive immune system on.
 • By stimulating this part of the innate immune system , we generate cytokines that
are able to activate APCs, promote production of antibodies, activate t cells and so
on.
• These are the response we want when we give the vaccine adjuvant

TLR4 recognises (lipopolysaccharide) lipid A

• LPS is the key component of the gram negative bacteria cell membrane. Embedded
in the other membrane.
• Lipid A anchor and polysaccharide.
• Effectly recognized by TLR4 (of the innate system).

MD2 binds to LPS lipid A

• TLR4 cannot bind directly to LPS. What is required is the intermediate molecule MD-
2. MD-2 binds to LPS, and then LPS-MD-2 complex that is bound to TLR-4.
• Lipid a tail of LPS is bound to MD-2 and then it is recognized by TLR4. When
recognized by TLR4, MYD88 and TRIF pathway is turned on, results in the production
of cytokines, chemokines and interferons which are required for the maturation of
the immune response.
• Molecular adjuvants try to switch these pathways on.
• How does the activation of the TLR pathway make a more productive immune
response.

TLR agonists (like LPS) are potent adjuvants

• They activate Myd88 and TRIF pathways
• Production of cytokines, chemokines, interferons
• These “shape” the adaptive response
• Because of these cytokines produced at the end of the TLR signalling pathways,
some of these are important for antibody production, some of them activate APC,
some of them activate t cells.
• These cytokines and chemokines shape the adaptive response.

How do these cytokines, chemokines and interferons shape the immune response ?

Bacterial (vaccine) proteins are processed by antigen presenting cells

• In general they act on APC. They make APC much more efficient at presenting
antigens.
• Take APC and expose it to some of these cytokines, it switches APC on, makes it
much more efficient at processing antigens and presenting them in MHC class 1 or 2.
Toll agonists

• Activate antigen presenting cells (like dendritic cells) to present antigen

• Cytokines produced activate CD4+ and CD8+ T-cells
• Switching the innate system on, might have 2 benefiical effects in term of antigen
processing.
• Activate APC
• Might activate CD4 and CD8 t cells.
• Could we use LPS as an adjuvant. Could we add LPS to a subunit vaccine? Would the
LPS switch the pathway on in APC, would it switch on signalling pathway, would it
result in the production of cytokines and chemokines which would lead to a much
better response to our vaccine antigen?
• IN response we can, but there is downsides.

Over-activation of the immune system during infectious disease

• Cytokine storm.
• LPS tremendously efficient activator of TLR signaling pathways.
• Sepsis -> consequence of overproduction of cytokines. Whole system goes into a
positive feedback loop, there is greater production of cytokines that feed the
immune response and the result is sepsis.
• Difficult to treat as it is difficult to get out of that loop.

LPS is a key mediator of sepsis caused by bacteria

• Toxicity is a consequence of TLR pathway activation
• IL1, IL6 and TNFα are key mediator
• Gram negative bacteria major cause of sepsis in this country ( triggered by LPS)
• So highly activated it goes in a positive feedback loop and hard to switch the
pathway off.
• LPS is potentially a great adjuvant but you don’t want any of these side effects.

adjuvant vs toxicity
• How to balance adjuvant effects of LPS (production of cytokines that have a mature
and effective immune response) and the toxcity (over stimulate immune system and
get detrimental effects) .
• Can’t balance it out. Very difficult to gauge the amount of LPS you might need to give
you the right sort of response against vaccine antigen without tipping the immune
system to give a autoimmune response.
• So therefore you cant use LPS as a vaccine adjuvant, as the risks are too big.
Can LPS lipid A be modified to retain the adjuvant effect but reduce toxicity?

• Try to modify LPS in some way to preserve activity and reduce toxicity.
• Lipid A causes the LPS toxicity. Bit that anchors LPS into the cell membrane.
• Lipid A recognized by TLR. Phosphate group is critical for binding and activity also.
• What would happen if this phosphate group is removed, how would it affect the
adjuvant properties and the immune stimulatory properties of LPS.
• If you remove phosphate group from LPS, you get this molecule called MPLA.
• MPLA generated by acid treatment of lipid A
• Looks identical apart from lacking the phosphate group. It reduces a lot of the
toxicity, but retain the adjuvant activity of LPS.
• Switches TLR signalling on, but not to the detriment of the host. No damaging side
effects.

Why does monophosphoryl lipid A behave differently?

• MPLA is still able to elicit adjuvant effects without toxicity of LPS.
• TLR-4 on the surface, binding to LPS or MPLA, signals through MYD88 and the TRIF
pathway, and they terminate in the production of these inflammatory mediators.
• Diagram summarizes when we look at signalling via these 2 pathways after they have
been exposed to LPS or to MPLA.
• Graphs show transcript of genes after exposure of cells to LPS and to MPLA.
• Given with model vaccine anitgen -> Ovalbumin
• MYD88 strong responses when treated with LPS, high amounts of IFNy, IL-6, serpine,
CXC1,
• When treated with ovalbumin and MPLA, there is little activation of the pathway of
MyD88.
• If we look at TRIF pathway
• -LPS - high activation of all these genes
• -MPLA – good activation of all these genes
• When exposed cells to LPS, both signalling pathways are activated
• When exposed just to MPLA, just TRIF pathway is activated
• Consequently there are different cytokines and chemokines produced, and what we
can achieve is the beneficial production cytokines and chemokines that are required
for adjuvanting without the production of cytokines and chemokines that are
required for toxicity

monophosphoryl lipid A activates the TRIF but not MyD88 signalling pathway
• Both can bind to TLR4, where Lipid A signals via both pathways
• MPLA only signals via TRIF pathways
• TRIF pathway -> T cell activation
• MyD88 -> NFkB, inflammation
• Preserve activity for adjuvanting and reduce toxicity.
 • Used quite widely in adjuvant subunit vaccines. Mix with protein of interest,
stimulates TRIF pathway in Innate immune system, production in chemokines and
cytokines and switch on adaptive system.
• Don’t exactly know which pathways end with what (IFN-B) in the TRIF pathway.
• Not simplistic, as there are cross talk.
Other TLR adjuvants
• Apply the same principle to other agonist of the TLR signalling pathway.
• Peptidoglycan, DNA also work well as TLR adjuvants to switch these pathways on.

Live and killed vaccines don’t require adjuvants why not ?

• Popular back then.

• Whole microorgnisms. Don’t need to use adjuvants.
• As they are self adjuvanting.
• As an entire microorgniams have the agonist of the TLR signalling pathway, they
have LPS, peptodiglycan, etc. and they are switching on the TLR pathway.
• They are delivery system in their own right for the vaccine antigen, they can target
the vaccine antigen to the APC, they can get into the cytosol, get the antigen into the
right site.
• Not very popular now as there are side effects.
• As there will be a over stimulated immune response, as they have all these TLR
receptor agonists (LPS, peptidolglycan, flagellin etc.), swithcing TLR pathways on, you
start to see some of these detrimental effects, such as sepsis, elevated
temperatures, inflammatory responses, pain at site of infection.
• Not just giving vaccine antigen, you are giving the combination of other molecules
which switch the innate system on, activate the TLR pathway and so on.
• Self adjuvanting, much much likely to cause detrimental effects.
• Whole reason why people are interested in sub-unit vaccines as you can start to use
things in a much more controlled way. Pick the bits you need to give you the right
responses. Adjuvant properties you need to give you the right response.

Do adjuvants cause the side effects of vaccines?

• A lot of people think they do.

• Lots of adjuvants in current use are based on aluminium salts. Concern that maybe it
is not a good thing to inject people with aluminium, because of the association with
various neurodegenerative diseases. Not much evidence, but theoretical issue.
• Another concern that one of the infleunza vaccine that was used to combat H1N1
strain of infleunza is now known to cause narcolepsy in some people. It is not
actually the vaccine antigen, it is the combination of the antigen and adjuvant
together which are responsible of these side effects.
• Lot more to be discovered.
• Adjuvants are important or subunit vaccines will not work.
• Need to understand at how they work at a molecular level.
Lecture 13 and 14 - monoclonal antibodies, cancer immunotherapy and molecular imaging

Polyclonal v. Monoclonal Antibodies

• Because most antigens carry many different epitopes, animals injected with a single
antigen will produce a complex mixture of antibodies, each made by a different
clone of B cells
• This antibody response is said to be polyclonal and will not discriminate between
different antigens
• To discriminate between different antigens, antibodies need to be identified that
bind to epitopes specific to individual antigens
• These are known as monoclonal antibodies

• Monoclonal antibodies (mAb or moAb) are antibodies that are made by identical
immune cells that are all clones of a unique parent cell.
• Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell
lineages within the body (whereas monoclonal antibodies come from a single cell
lineage).
• If you want to use antibodies to target specific antigen you have an issue. If you take
an antigen and put it in the mammalian host (rat or mouse usually), all that happens
is that a lot of b cells being switched on all producing their own specific antibody
recognizing different epitopes of the antigen you have immunized with -> that is a
polyclonal antibody. You have a pool of antibody. Might be 1 b cells that is producing
antibody of interest but rest is recognizing epitopes that are conserved for other
antigens.
• Monoclonal antibodies is you can identify the one specific b cell in that whole b cell
response that is producing a specific antibody of interest. Recognizing the epitope
that is highly speciifc for the antigen of interest that doesn’t cross react with
conserved epitopes on different antigens.
• Need to find the B cell that is producing its single antibody. Known as monoclonal
antibody. Each individual b cell is producing its own monoclonal antibody.