Immunoevasion and Immunotherapy in Infectious Disease Flashcards
after more than 70 years of widespread use, evolution of disease-causing microbes has resulted in…
… many antimicriobials losing their effectiveness
As microbes evolve, they adapt to
their environment
If something stops them growing (e.g. antimicrobial), it introduces
a selection pressure
Genetic mutations which counter the selection pressure will be reproduced in
offspring and fixed in the population.
New mechanisms to resist the antimicrobials will
evolve.
in the 1970s Hungary was highly dependent on
penicillin for treating infections.
in the 1970s Hungary was highly dependent on penicillin for treating infections.
Quickly this led to
half of the strains of Streptococcus pneumoniae being resistant
- Things improved only when penicillin usage dropped
Antibiotic resistance is a global problem;
increasing numbers of bacteria are
becoming resistant to antibiotics
It is predicted that by 2050
AMR infections will be leading ahead of cancer as causes of death.
And we have been being given cautionary notes ever since 1945, even by the discoverer of penicillin himself…
what is PBP?
penicillin binding protein (transpeptidase involved in peptidoglycan cell wall synthesis)
what kind of antibiotic is methicillin?
a β-lactam antibiotic (penicillin class)
β-lactam antibiotics are effective against
gram +ve bacteria because they inhibit the trans-peptidase involved in cell wall synthesis
Bacteria develop resistance through
acquisition of β-lactamase gene
β-lactamase hydrolyses
the β-lactam ring – drug becomes ineffective
Staphylococcus aureus
A gram +ve commensal bacteria, part of skin and
nasopharangeal flora – normally causes
no harm
Infections can occur when
barriers are breached (skin and mucosal barriers)
Staphylococcus aureus
A gram +ve commensal bacteria, part of skin and
nasopharangeal flora – normally causes no harm
Infections can occur when barriers are breached
(skin and mucosal barriers)
can treat with
β-lactam antibiotics (methicillin)
- methicillin resistant strains (MRSA)
MRSA stands for…..
… methicillin-resistant staphylococcus aureus. This type of bacteria causes staph infections that are resistant to treatment with usual antibiotics.
mrsa occurs most frequently…
… among patients who undergo invasive medical proceuures or who have weakned immune systems and are being treated in hospitals and healthcare fcacilities such as nursing homes and dialysis centers
mrsa in healthcare settings commonly causes
serious and potentially life threatening infections such as bloodstream infections, surgical site infections, or pneumonia.
Disease incidence caused by MRSA in UK and Europe has been on a steady increase since
1993
Recent years has seen a fall in nosocomial MRSA infections in the UK, due to
the introduction of 60% isopropylalcohol gels in wards (spread has been limited)
However MRSA is still there and remains a global problem with strains now
carrying additional vancomycin resistant genes (multi-drug resistant MRSA strains)
Vancomycin is an antibiotic of
the glycopeptide class
Like the β-lactams, glycopeptide antibiotics target
cell wall synthesis and are thus most effective against gram +ve – bind to; D-Ala-D-Ala in peptidoglycan
Vancomycin is used to treat MRSA – but
vancomycin resistance is becoming a problem
mid-1980’s was an area which
saw the greatest development of new antibiotics
during the 2008 - 2012 period only
2 new antibiotics were developed
(running out of targets??)
during the 2008 - 2012 period only 2 new antibiotics were developed
(running out of targets??)
this accounts for the apocalypse hysteria
so what is being done?
immunotherapy is one avenue that is being explored
multidrug resistant Tuberculosis (MDR-TB) – first new antibiotic to be discovered for
30 years (2015)
when is immunotherapy important?
particularly important for viral, chronic or difficult to treat infections such as; methicillin resistant Staphylococcus aureus (MRSA)
MRSA can enter the blood causing…
…septicaemia or sepsis (ultimately death)- systemic inflammation
post antibiotic era looking to establish
new ways to treat antibiotic resistant bacteria; including MRSA
Immunotherapeutic strategies examples:
-immunise (some epitopes identified); preventative
-cytokine treatment, to boost immune response
-adoptive immunoglobulin’s, pathogen specific antibody therapy
-adoptive T cell transfer (donor/self pathogen specific T cells)
what provides the first line of defence?
Anatomical barriers SKIN and MUCOSA provide the first line of defence against microbial infection (innate)
Biochemical secretions have
an ‘antimicrobial’ activity
Human 1-defensin is a
polypeptide with positively charged and hydrophobic regions, that creates pores in the membrane
positive domain is attracted to
negative phospholipid
hydrophobic domains group
together - creating pore
lysozyme hydrolyses
NAG-NAM glycosidic bonds (gram +ve bacteria)
go look at 34
how do lysozymes break down bacteria?
1) rupture of peptidoglycan by lysozyme action
2) inflow of water
3) lysis
A newly synthesized PG monomer consists of a
disaccharide, NAG linked to NAM with an
attached peptide stem, and the NAM is anchored to the membrane via a lipid carrier
Lysozyme hydrolyzes the
β-1,4 glycosidic bond between the NAG of 1 monomer and the NAM of the adjacent monomer
Lysozyme hydrolysis of PG leads to…
…cell wall instability and bacterial cell death.
Lysozyme can also kill bacteria independently of
PG hydrolysis
Lysozyme can also kill bacteria independently of PG hydrolysis - mechanism involving its
cationic nature
Cationic killing of bacteria may involve
the formation of pores by lysozyme
(red cylinders) on the bacterial cell membrane.
Oat-A promotes
acetylation at the N-muramic acid of peptidoglycan
describe the inflammatory response driven by leucocytes?
tissue macrophages become activated and send chemotactic signals (cytokines) to recruit neutrophils (phagocytic leukocytes)
cytokines are important…
… mediators
Cytokines are important mediators in the inflammatory response and neutrophils (50 – 70% of total leukocyte count) are…
… important cells for recognising and killing bacteria (phagocytosis, cytotoxicity and cytokine secretion)
neutrophils make up
50 to 70% of all leukocytes
Neutrophils: 50 to 70% of all leukocytes, important for
bacterial and fungal infections, last only hours to a few days in circulation
function of neutrophils
Phagocytic function (engulf and digest bacteria)
how do neutrophils identify bacteria?
Identify bacteria through Pathogen Associated Molecular Pattern (PAMP) receptors – toll-like receptors (TLRs)
neutrophils are […] in functioin?
These cells are innate in function (see all that is foreign ‘non-self’ and kill)
How many TLRs are identified?
12
what does TLR2 do?
recognises peptidoglycan of gram +ve bacteria cell wall (e.g. MRSA)
what does TLR4 do?
recognises lipopolysaccharide of gram -ve bacteria (e.g. Salmonella)
Cytokines are chemical (peptides) that bind
cognate receptors on immune cells
Proinflammatory cytokines activate
immune cells and mediate inflammation by targeting vessel endothelial cells
what are 4 types of cytokine?
interleukin-1B (IL-1B)
IL-18
Interferon-a (IFNa)
Tumour necrosis factor-a (TNFa)
interleukin-1B (IL-1B) immune target cell ?
Macrophage, monocyte & neutrophil
interleukin-1B (IL-1B) major actions?
Activates cells, stimulates inflammatory cytokine release
interleukin-1B (IL-1B) major outcomes?
Acute phase response, fever, wound healing & pain
IL-18 immune cell target?
Macrophage & neutrophil
IL-18 Major actions?
T cell maturation, stimulates IFN production
IL-18 major outcomes?
Regulates homeostasis
Interferon-α (IFNα) immune cell target?
Macrophage and NK cells
Interferon-α (IFNα) major actions?
Activates macrophages and NK cells
Interferon-α (IFNα) major outcomes?
Fever (flu-like symptoms)
Tumour necrosis factor-α (TNFα) immune cell target?
Macrophage, Th1 cells and NK cells
Tumour necrosis factor-α (TNFα) major actions?
Potent immune cell activator
Tumour necrosis factor-α (TNFα) major outcomes?
Acute phase response, fever & sepsis
Cytokine therapy potential for
treating antibiotic resistant infection
Cytokines have a central role in
driving the immune response
Cytokines have a central role in driving the immune response, thus administering certain cytokines has the potential to …
… improve the host immune response during MRSA infection
in this study IL-18 (important in inflammatory immune response), protected from…
… MRSA induced death, following burn injury
IL-18 was found to induce
significantly neutrophil number and function (doubled)
IL-18 administration correlated with
a decrease in blood associated MRSA sepsis
what does antibody therapy involve?
involves the generation of ‘humanised’ monoclonal antibodies (anti-bodies that recognise specific bacterial epitopes)
in antibody therapy, spleen cells are harvested from
mice challenged with the epitope
Spleen cells harvested from mice challenged with the epitope are used to form
hybridoma culture with myeloma cells (plasma cell cancer)
Bacteria epitope specific antibodies are
harvested from the culture medium
The antibodies are then transfused into
a patient who has been diagnosed with bacteria infection (e.g. MRSA)
describe antibody structure?
‘Y’ shaped proteins that consist of two heavy chains and two light chains held together by intermolecular disulphide bonds
what are the variable domains (V) of antibodies for?
antibody specificity
what are the constant domains (C) of antibodies for?
biological activity
FAB (variable end) binds to
the antigen (bacteria or foreign invader)
describe antibody bacteria binding
1) Antibody binds to bacterium
2) Antibody-coated bacterium binds to Fc receptors on cell surface
3) Macrophage membrane surrounds bacterium
4) Macrophage membranes fuse, creating a membrane bounded vesicle, the phagosome
5) Lysosome fuse with the phagosome, creating the phagolysosome.
Antibodies also activate the
complement system
Antibodies also activate the complement system; specialised proteins that collectively form the
‘membrane attack complex’ – produce holes in bacteria cell membrane
Antibodies specific to the pathogen bind at
the FAB region
The protruding FC region contains
complement binding sites
The protruding FC region contains complement binding sites that
bind and activate the complement system sequentially (C1 to C9)
complement proteins arrange in
a specific way (and order) to create pores in the pathogen membrane
complement proteins arrange in a specific way (and order) to create pores in the pathogen membrane - a process known as
the complement cascade
Unlike neutrophils (innate cells that are activated towards foreign antigen indiscriminately) B cells (a type of lymphocyte) are
activated to specific regions of the antigen (foreign invader) ‘epitopes’ – ADAPTIVE IMMUNE CELLS
Each B cell has a
unique receptor
Each B cell has a unique receptor – once activated
differentiates into plasma cell that secretes antibody (same shape as receptor)
Create transgenic humanised B cells toward a
specific bacterial epitope
Isolate cells and create
hybridomas (immortalised antibody secreting cells)
Purify antibodies from
supernatant and transfuse
the antibodies are then transfused into a patient who
has been diagnosed with bacteria infection (e.g. MRSA)
Antibodies can be designed to target
bacterial ‘epitopes’ at numerous levels
Potential bacterial epitope targets:
Target 1 - antibodies directed to surface increasing phagocytosis (opsonising)
Target 2 - antibodies directed to virulence factors such as toxins (neutralising)
Target 3 - antibodies directed to virulence factors such as quorum sensing peptides (sequestering)
describe opsonising
Opsonises specific antibiotic resistant bacteria; tagging them for phagocytic destruction
different antibiotic resistant bacteria epitopes are under investigation; including
the bacteria cell surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG)
promising results highlighted the efficacy of these antibodies to kill
various multidrug resistant bacteria, via opsonophagocytosis
antibodies were able to prevent death by
peritonitis in pre-clinical models, even when co-infected with MRSA
enterotoxin-B is a
virulence factor produced by some MRSA strains
enterotoxin-B is a virulence factor produced by some MRSA strains
antibodies have been designed to recognise this pathogen and have been shown to
improve survival in pre-clinical MRSA-sepsis models
vaccination with enterotoxin-B epitope provided
immunity to infection
antibody treatment also reduced
the amount of MRSA present in skin lesions
antibody treatment also reduced the amount of MRSA present in skin lesions
thus treatment could have wider implications for
topical MRSA infections (such as post-operative infections).
MRSA establishes virulence though
quorum sensing
since MRSA establishes virulence though quorum sensing, antibodies have been designed to
target this process
antibodies have been designed to target
quorum sensing peptides
antibodies have been designed to target quorum sensing peptides; such as
AIP-4
the sequestering (mopping-up) of AIP-4 by antibodies, inhibits
quorum sensing-mediated gene expression changes
the sequestering (mopping-up) of AIP-4 by antibodies, inhibits quorum sensing-mediated gene expression changes -> what does this cause?
decreases MRSA virulence -> protects pre-clinical models from a lethal S. aureus dose
Like B cells, T cells are
adaptive (Specific)
Like B cells, T cells are adaptive (specific) and activated through
unique cell surface receptors
T cells are
lymphocytes (like B cells)
T cells are lymphocytes (like B cells) but do not
secrete antibodies
Two types of T cell:
1) Cytotoxic T cells (or Tc cells) - viral infections
2) Helper T cells (or TH cells)
how does adoptive t cell therapy work?
take autologous or HLA matched donor t cells, before expanding the pathogen specfic cells in vitro and infusing back into the patient
adoptive t cell therapy has the potential to…
… genetically engineer t cells before infusion.
adoptive t cell therapy is excellent for
viral infections, where viral specific cytotoxic t cells can be generated
Hepatitis B virus causes
causes infection of the liver
Hepatitis B virus causes infection of the liver and is associated with the onset of
cirrhosis (liver scarring) and hepatocellular carcinoma
Adoptive transfer of HBV specific donor t cells neutralised
infection in patients with chronic HBV infection
Epstein-barr virus (EBV) is associated with
lymphoproliferative disorders and malignancies, such as; lymphoma and chronic lymphocytic leukaemia (CLL)
EBV infected B cells can become
malignant
EBV infected B cells can become malignant, with affected cells expressing
EBV specific mRNA and viral proteins – viral antigen
Numerous studies show that adoptive transfer of EBV specific T cells can enhance
anti-tumour response and improve survival in pre-clinical disease models
Adoptive transfer in conjunction with cytokine (IL-15), may enhance
the effects by relieving immune suppression (combination immune therapy)
L. monocytogenes is one of the most
virulent food born pathogens, with death resulting in up to 30% clinical infections
L. monocytogenes specific cytotoxic t cells neutralise
infection
various L. monocytogenes epitopes have
been investigated
combining more than 1 epitope specific cytotoxic t cell clone in mixed culture assays improved
the efficacy of L. mono-cytogenes killing
Bacteria have evolved several mechanisms to
‘evade’ the immune response
Viruses present another challenge:
All immune cells ignore
self antigen (some immune cells like T and B cells are educated to ignore self)
But viruses replicate inside
our cells (hide away from neutrophils)
How does our immune system deal with viral infected cells?
major histocompatibility complex (MHC) molecules present vial peptide on cell surface
MHC-I is expressed on
all nucleated cells and presents antigen (viral peptides, cancer peptides) to cytotoxic T cells (Tc cells)
If a Tc cell has a receptor complementary to the viral antigen it will become
activated
If a Tc cell has a receptor complementary to the viral antigen it will become activated
Tc cells will then
destroy the viral infected cell (not by phagocytosis) but through the release of cytotoxic granules.
Like bacteria, certain viral strains have
evolved / acquired genes
Like bacteria, certain viral strains have evolved / acquired genes that when translated into protein, …
… help the viral evade Tc cells during the intracellular replication phase
what are two strategies used by viral strains to evade Tc cells ?
1) Viral homologues to human cell surface immunosuppressive molecules
2) Viral homologues to human immunosuppressive cytokines
It is important to prevent immune responses in certain parts of the body in order to…
… protect certain vital organs from immune destruction (immune privileged sites
what proteins protect cells from immune attack in immune privileged sites?
Cell surface proteins (e.g. CD200, PD-1), protect these cells from immune attack
what is CD200 ?
CD200 is a cell surface immunoprotective glycoprotein – expression high in immune privileged sites
Downregulation of CD200 is linked with
the development of neurodegeneration (multiple sclerosis)
cancer cells that up-regulate CD200 are
protected from immune attack
Study found that herpesvirus K14 protein (40% homology) mimics
the effects of CD200 by interacting with the CD200 receptor (CD200R), supressing immune cell activity and the release of proinflammatory cytokines
Study found that herpesvirus K14 protein (40% homology) mimics the effects of CD200 by interacting with the CD200 receptor (CD200R), supressing immune cell activity and the release of proinflammatory cytokines
This virus is linked to …
… the development of Kaposi’s sarcoma
Viral homologues for CD200 found in
poxviruses and cytomegalovirus (CMV)
Viral K14 protein binds to
CD200R with similar affinity and kinetics as CD200
K14 mediates
immunosuppression in immortalised cells
As well a lifting the immuno-suppression mediated by CD200 through the receptor, certain antibody clones have been shown to
promote Antibody Dependent Cell Cytotoxicity (ADCC) – improve Tc cell activity (tags cells for destruction)
Proinflammatory cytokine production is a vicious cycle, which must be
attenuated (homeostatic feedback)
Production of immunosuppressive cytokines can provide
feedback control – e.g. interleukin-10 (IL-10)
what is IL-10 ?
IL-10 is a peptide secreted by special immunosuppressive cells known as regulatory T cells (Treg)
IL-10 is a potent
immunosuppressive cytokine
IL-10 is a potent immunosuppressive cytokine:
- supress inflammation
- supress activation
- supress proliferation
- supress chemotaxis
- anti-pyrogenic
Human herpes virus-4 (aka EBV) IL-10 homologue induces
immune suppression
Viral homologues of IL-10 are
old news
Epstein-Barr virus (EBV IL-10) homologue further
characterised
EBV linked to
lymphoma and other cancers
