Module 5 Flashcards
tools used in order to understand and help fight infections
-ELISA
-Flow Cytometry
-Monoclonal Antidies
ELISA
Enzyme-linked immunosorbent assay (ELISA) is a fundamental tool of clinical immunology based on the principle of antigen-antibody interaction
Flow Cytometry
method of detecting and quantifying different cell types in a mixed cell suspension.
analyzes cell suspensions and translates this information into datasets
Monoclonal Antibodies
developed by Georges Kohler and Cesar Milstein in 1975
-antibodies that are produced by a single clone of a B cell that are specific for a single epitope
ELISA can be modified to detect:
pepties, proteins, antibodies, hormones and other molecules
why is ELISA highly specific?
based on principle of antibody-antigen interaction an the antibody binding site is very specific to one antigen
1st step of indirect ELISA
bottom of wells are coated with an antigen that is specifically recognized by the antibody you wish to measure (primary antibody)
What happens between steps in indirect ELISA
wells are washed to remove any excess antigen not attached to the bottom of the well
step 2 of indirect ELISA
sample containing antibody to be measured is added to well. Primary antibodies, if present, will bind to the antigens attached to the bottom of the well
Step 3 of indirect ELISA
an enzyme-conjuated secondary antibody is added to the well. THis secondary antibody will bind to the Fc portion of the primary antibodies already presnt in the well. The secondary antibodie used specifically recognizes antibodies from a particular animal
step 4 indirect ELISA
the substarte of the enzyme attached to teh secondary antibody is added to the well. The reaction of the substrate (a chromogen) and the enzyme produces a coloured product which can be measured by absorbance
chromogen
a substance that can be readily converted into a dye or other coloured compound
What does ELISA measure
- a coloured reaction product by absorbance with the help of a machine
Indirect ELISA
detects or quantifies antibody. Ex, to determine the presence of serum antibodies against HIV
Enzyme-conjugated secondary antibody
a secondary antibody specifically binds to the primary antibody. (has enzyme attached to it)
how can flow cytometry be used to diagnose AIDS/HIV
used to identify and count these specific T-cells in a blood sample
How does Flow cytometry work
-single file stream of cells passed through laser light
-scattering of laser light unique to each cell type
-measuring FSC allows for discrimination of cells by size
-FSC intesnsity proportional to diameter of cell
-SSC provides infro about internal complexity of cell
-also used to det. proportion of cells expressing a particular antigen via stain
FSC and SSC measured in conjunction
allow for detection of specific heterogenous population
What does flow Cytometry measure
-physical properties of a cell
-detecg specific antigens on or inside a cell
what is readily determined via flow cytometry
-total number of cells in suspension
-number of cells of a particular type in the suspension
-overall composition of suspention
what is flow cytometry used to determine
complete blood counts
when would one use flow cytommetry
used to count and analyze size, shape and properties of individual cells within a heterogenous population of cells
Aneuploidy
the presence of an abnormal number of chromosomes
Main diagnostic test of flow cytometry used to diagnose cancer
detecting DNA sneuploidy, analyzing cell cycles, and th e immunophenotypical characterization
what substances is flow cytometry used on
mainly blood, also bodily fluids or emulsified tissues
How do monoclonal antibodies work?
monoclonal antibodies are produced in the lab by hybridomas, imortal cells that produce unlimited quantities of one identical antibody
Hybridomas
hybridomas are the result of fusion bw plasma cell and cancerous cell (share properties of both)
Properties of Myeloma cell
immortal growth that divides indefinitely
properties of plasma cell
produce specific antibodies against one antigen
properties of hybridoma cell
a perpetual source of antibodies against one antigen
what do monoclonal antibodies measure?
-not a specific technique of measurement
-clinical application include immunotoxins and radiolabelled antibodies
Immunotoxins
consist of tumour specific monoclonal antibody attached to deadly toxin
long term objective of immunotoxins
to target and eliminate tumour cells and treat cancer
Radioleballed antibodies
Monoclonal antibodies tagged with a radioactive isotope can be used to diagnose tumour earlier than other methods. Radiolabelled antibodies can bind to antigens on a tumour thereby allowing the percise location of a tumour within the body to be visualized
what is a vaccine
biological preparation providing articfical active immunity to particular disease-causing agent
types of vaccines
- Live attenuated vaccine
- Kill-inactivated vaccine
- Toxoid Vaccine
- Subunit vaccine
Characteristics of live attenuated vaccine
-contains modified strain of disease-causing agent which has lost pathogenic ability but retains its capacity to replicate within host
examples of live attenuated vaccine
smallpox
-oral polliovirus
-measles
advantages of live attenuated vaccine
-provides prolonged exposure to the disease-causing agent, and its suitable to generate cell mediated immunity
disadvantages of live-attenuated vaccine
-potential to revert to virulent form
-requires specific storage and transport conditions
characteristics of killed-inactivated vaccine
-contains strain of the disease-causing agent that has been inactivated by heat, chemicals or radiation
-ability to generate an immune response but is unable to replicate
examples of killed-inactivated vaccine
-rabies
-Flu (influenza) vaccine
advantages of killed-inactivated vaccine
-safer as i cannot mutate to virulent form
-easy to store and transport
disadvantages of killed-inactivated vaccine
-generally requries multiple booster doses to maintain immunity
-must be administered by injection
characteristics of toxoid vaccines
-contains inactivated toxin which is a product from the pathogen that is causing the disease
examples of toxoid vaccines
-tetanus
-diptheria
advantages of toxoid vaccine
-safe as it is not a living organism that can divide, spread and/or revert
-stable as they are less susceptible to changes in temperature, humidity and light
disadvantage toxoid vaccicne
-may require several doses and usually need an adjuvant (substance enhancing bodys immune response to antigen)
characteristics of subunit vaccine
contains only small part or fragment of disease causing agent
examples of subunit vaccine
-hepatitis B
advantages of subunit vaccine
-safest type of vaccine, can be used on everyone, including immunocompromised, pregnent and elderly
disadvantage of subunit vaccine
-rarely successful at inducing long lasting immunity, which means it will require multiple booster doses to maintain immunity and might even need to be conjugated to a carrier
why are there so many types of vaccine
-not one size fits all approach
-varies with caracteristics of pathogens and the way the IS recognizes them
-objective remains the same: develop effective immune response and produce immunological memory so the body will be able to counter the fully active form
mRNA vaccines
-most recent vaccine that has changed the field
-used in several formulations
-investigates for other infectious and non infectious conditions
examples of mRNA vaccine
-COVID 19
-SARS CoV 2
how mRNA vaccines work
use mRNA to produce viral proteins and recruit immune cells to respond to the antigenic target. proteins are then displayed on surface of antigen presenting cells to induce B cells and T cell immunity
RNA and DNA based vaccines
involve making genetic material only; do not requier use of whole virus
mechanisms for mRNA vaccine for COVID 19
- Vaccine production
- Host cell
- APC
- Immune Response
1st mechanism for mRMA vaccine for COVID
Vaccine production
-mRNA made in lab from DNA
-mRNA encodes antigen of the virus
-mRNA incoroporated into a formulation that is administered as vaccine
2nd mechanism for mRMA vaccine for COVID
Host Cell
-once inside the body, the mRNA enters the host cell machinery to produce protein
3rd mechanism for mRMA vaccine for COVID
APC
-the newly formed protein exits cell and is recognized by APC
–APC internalizes protein and processes it into peptide (antigen)
-APC then displays antigen on surface via MHC complex
4th mechanism for mRMA vaccine for COVID
IMMUNE RESPONSE
-antigen is recognized by T helper cell initiating immune response
-B cells produce antibodies that stop virus from infecting cells
-T cells destroy cells infected with virus
Antiviral medications
-taken orally and does not prevent infection, treats it once you are already positive
two types of antiviral mediations
- Polymerase inhibitor
- Protease Inhibitor
Polymerase inhibitor
-first antiviral medication
-polymerase is an enzyme that plays a central role in viral replication and transmission
type of polymerase inhibitor
Molnupiravir
-used to treat COVID 19
-increases frequency of viral RNA mutations and impairs replication of virus
Protease Inhibitor
-second antiviral medication
-cut proteins into smaller, more workable pieces
-administered in combination
example of protease inhibitor
Nirmatrelvir
-stops protease from cutting viral proteins into functional pieces
Ritonavir
-protects nirmatrelvir from destruction by the body and allows it to keep working
-these drugs disrupt assembly of virus so they cant replicate and infect other cells
Evolution of vaccines
-recognized by british medical journal as one of greatest medical advances in history
Edward Jenner
use of extracts from cowpox lesions to protect patients against smallpox
HPV VLP Vaccines
-virus like particle vaccine with 100% efficacy
-VPLs composed of structural proteins of HPV which can self assemble into particles that resemble the natural virus
-NOT INFECTIOUS (dont contain DNA)
-currently 3 authorized for use in canada
-
Ebola Virus
-vaccine developed using glycoproteins from EBOV and putting them into a live attenuated recombiant VSV that expresses transmembrane glycoproteins of EBOV and MARV
-vaccine found to be 100% effective against EBOV
Genital Herpes Vaccine
-subunit vaccine developed composed of structural protein of HSV 2
-first shows high efficacy then low efficaacy on larger populaiton
-consider for developing life attenuated vaccine for genital herpes
phases of vaccine development
- Lab studies
- Preclinical (mice)
- Clinical phase I (small pop)
- Clinical phase 2 (bigger pop)
- Clinical phase 3 (large pop)
- Health canada approval
1 phase of vaccine development: Lab studies
-identify infectious agent causing the disease and select strain
-screening to identify suitable antigen
-developing and testing the manufacturing process
2 phase of vaccine development: Preclinical
-research carried out in animal models
-demonstrate immunogenicity of vaccine
-carry out safety studies to evaluate possible toxicity
3 phase of vaccine development: Clinical phase 1
-small % of vaccines progress to here
-small scale trials in humans (10-100)
-assess safety by evaluating rxns
-provides preliminary data on immunogenicity and the response it evokes
4 phase of vaccine development: clinical phase 2
-bigger scale (50-500)
-collect data on safety, side effects, and efficacy
-evaluates dosage requirements of vaccine
-define optimal dose and schedule (ie boosters)
5 phase of vaccine development: Clinical phase 3
-multiple geographic sites
-hundreds/thousands of subjects
–evaluate efficacy under natural disease conditions
–researchers required to demonstrate efficacy in target populations and complete safety assessment
–apply regulatory authorities for a liscence to market
6 phase vaccine development: Health canada approval
-candidates submitted to be considered for approval with evidence
Vaccination in Canada
-routine
-provided free
-critical role in heard immunity
what vaccine do we get every fall
-influenza
challenges associated with vaccines
-cost
-the cold chain (storage and transportation, often needs refridgeration)
-continuous monitoring (if disease causing agents evolve no longer effective)
-the gold standard (new vaccines must be as good or better than the last)
two important structures of influenza virus
Neuroaminidase Antigen & Haemagglutinin Antigen
Neuroaminidase Antigen
-surface protein that removes sialic acid from cell surfaces and enables new viral copies to infect and spread to other cells
-11 subtypes
Haemagglutinin Antigen
surface protein that recognizes and binds sialic acid on cell surface glycoproteins. HA-cell surface interactions lead to endocytosis of thevirus and the HAs are activated to fuse the endosome and viral membrane
-18 subtypes
How many potential strains of influenza virus
11 NA x 18 HA = 198 potential strains
Heard immunity
form of indirect protection from an infectious disease that occurs when a large proportion of the population is immune to an infectious agent
why is heard immunity beneficial
provides protection for individuals who are not immunized
how can heard immunity be measured
by calculating basic reproduction number (R nought) and this determines heard immunity threshold. As R nought increases, higher immunization coverage is required to achieve heard immunity
R nought
average number of secondary cases in an infectious disease arising from a typical case in totally susceptible population
what is cancer characterized by
an error in cell growth or death
difference between cancer cells and normal cells
-cancer cells do not need specific growth factors to divide
-cancer cells dont response to signals that cause normal cells to stop dividing
cancer cells
-self cells that change to escape normal growth-regulating mechanisms
-changes are result of DNA alterations inducing cell transformation
-usually result of carcinogens or radiation
what is a tumour
abnormal mass of tissue due to cancer cells dividing and growing
cancer immunotherapies
aimed at enhancing host anti-tumour immune responses based on secreted or cellular components of the immune system
types of tumours
benign or malignant
benign tumour
not cancerous, unable to grow indefinitely or invade surrounding tissues
Malignant tumour
-cancerous and has the ability to metastasize
-become progressively invasive
Metastasis
the colonization by tumour cells of sites different from their primary site of origin. Small clusters will break off and invade surrounding blood or lymph vessels travelling through the body to proliferate
characteristic of cancer cells
resilience - they cope with disturbance well
where does biomedical research of cancer originate
a line of cancerous cells known as HeLa cells
Henrietta Lacks
samples of ovarian cells were taken and first ever cells to survive outside of the human body
cancer immunity cycle
- Release of cancer cell antigens
- Cancer Antigen presentation
- Priming and activation
- Trafficking of T - cells to tumours
- Infiltration of T-cells into tumours
- Recognition of cancer cells by T cells
- Killing of cancer cells
step 1 of cancer immunity cycle
Release of cancer cell antigens (cancer cell death)
-antigens released by mutated cancer cells, indicating they are not healthy. IS recognizes these antigens
step 2 of cancer immunity cycle
Cancer antigen presentation (dendritic cells/APCs)
-cells of IS capture released antigens and travel to lymph where they find T cells
Step 3 of cancer immunity cycle
Priming and activation (APCs & T cells)
-T cells activated by antigens and the immune repsonse is initiated
Step4 of cancer immunity cycle
Trafficking of T cells to tumours (CTLs)
The activated T-cells move through the blood vessels to the site of the tumour
step 5 of cancer immunity cycle
Infiltration of T-cells into tumours (CTLs, endothelial cells)
-once the T cells reach the cancerous cells, they invade the tumour to attack it
step 6 of cancer immunity cycle
recognition of cancer cells by T cells
-T cells recognize cancer cells because if the antigens they had previously released
step 7 of cancer immunity cycle
killing of cancer cells (immune & cancer cells )
-T-cells initiate a pathway that results in cancer cell death
Immunosurveillance
-tumour cells are identified and kept under control
Immunoediting
-connection between tumour cells and IS
-how cancer cells overcome IS to form tumours
consitiuated by three phsases:
1. Elimination
2. Equilibrium
3. Escape
Phase 1 of immunoediting
ELIMINATION
-IS quickly reacts to remove tumour cell
-variety of immune cells including NK cells, cytotoxic T cells, & helper T cells can recognize and work to eliminate
Phase 2 of immuniediting
EQUILIBRIUM
-if tumour cells not eliminated, they can enter a stste of equilibrium where proliferation is matched by cell death by IS
-can last for short time or many years
Phase 3 of immunoediting
ESCAPE
-tumour cells no longer recognized by IS and avoid elimination so they are able to grow and proliferate into a tumour
Cancer evasion of immune response via reduce MHC expression
-tumour cells display low levels of MHC class I molecules on surface
-as cytotoxic T lymphocytes recognize antigens and absense of molecules will inhibit recognition of tumour cells
Cancer evasion of the immune system via poor cosimulatory molecules
-T cells require both expression of MHCand cosimulatroy molecules to become activated
-tumour cells lack cosimulatroy molecules which contribute to poor immunogenicity so T cells are only partly activated
WHy is immunotherapy potential to be highly effective in treating cancer
-able to attack cancerous cells throughout all organs in the body
-allows IS to specifically target and eliminate cancer cells without damging healthy cells resulting in fewer side effects
-takes advantage of immunological memory, allowing for the possibility of long term protection
-applied to almost all types of cancer
most effective population in killing cancer cells
T cells
Tumour infiltrating lymphocytes
B cell lymphocytes that are prognostic biomarkers in some cancers (predict course of disease)
how do tumour infiltrating lymphocytes work
leavethe blood and migrate to infiltrate the tumour under influence of various chemotatic gradients of specific types. can be a mix of B and T cells, NK & dendritic cells,sometimes macrophages.
patient prognsis with TILs
improved
T cell inflammed tumour
solid tumours
t cell inflammed (hot) or T cell non inflammed (cold)
T cell non-inflammed tumours
-cold tumours
-lower numbers of CD8+ TILs
-low levels of interferon genes
-inferior response to treatment
conversion of cold to hot
one could convert cold to hot by stimulating tumour interferon activity
Immunoscore
-new reliable prognostic biomarker for cancer diagnosis
-measures density/numbers of T-cells in the center and periphery of tumour
-help stratify having high or low risk cancers
