Module 5

ELISA

Enzyme-linked immunosorbent assay
Detect and quantify substances
Antibody bind specific antigen epitope

ELISA: Measure

Coloured product absorbance with spectrophotometer
Detect antibody/antigen/disease presence

Sandwich ELISA

For antigens

Indirect ELISA

For antibodies

Indirect ELISA 1: Coat Wells

Antigen on well bottom
Recognize by primary antibody (measured)
Wash wells

Indirect ELISA 2: Add Sample

Primary antibodies bind antigens
Wash wells

Indirect ELISA 3: Add Secondary Antibody

Enzyme-conjugated
Bind Fc portion of primary antibody
Recognize animal antibodies (anti-human)
Wash wells

Indirect ELISA 4: Add Substrate

Chemogen: Substance converting to dye
React with antibody enzyme
Produce coloured product
Measure absorbance

Flow Cytometry

Detect and quantify immune cell in mixed suspension

Flow Cytometry: Measure

Physical cell properties
Antigens on cell, total cells, number of specific cells, complete blood counts

Cell Type Flow Cytometry 1: Laser

Single file cells pass through laser
Unique light scatter from cell size

Cell Type Flow Cytometry 2: Analyze Light Scatter

Forward Scatter (FSC): Intensity proportional to cell diameter
Side Scatter (SSC): Internal granules

Cell Proportion Flow Cytometry 1: Label Cells

Cell-specific antibodies
Stain with fluorescent marker

Cell Proportion Flow Cytometry 2: Light

Cells pass through light
Excite marker
Emit light at different wavelengths

Flow Cytometry: HIV/AIDS Diagnosis

Count T-cells

Flow Cytometry: Cancer Diagnosis

Detect DNA aneuploidy (abnormal chromosomes)
Analyze cell cycles for immunophenotypical characterization

Monoclonal Antibodies

Antibodies from 1 B-cell clone (homogenous)
Epitope-specific

Monoclonal Antibodies: Immunotoxins

Antibodies attach to toxin
Target and eliminate tumour cells

Monoclonal Antibodies: Radiolabelled Antibodies

Tag antibodies with radioactive isotope
Locate and visualize tumour antigens

Monoclonal Antibodies 1: Hybridomas

Fuse plasma cell and cancerous (myeloma) cell

Monoclonal Antibodies 2: Antibodies

Plasma Cell: Produce specific antibodies
Myeloma Cell: Indefinite growth and division
Hybridoma Cell: Indefinite identical antibody production

Monoclonal Antibodies: Cancer Treatment

Target specific cancer cells

Vaccines

Contain modified pathogen
Produce immunological memory

Live-Attenuated Vaccine

Modified pathogen strain
No pathogenic ability
Replicate in host

Live-Attenuated Advantages

Prolonged pathogen exposure
Generate cell-mediated immunity

Live-Attenuated Disadvantages

Revert to virulent form
Not for immunocompromised and pregnant
Specific storage and transport

Live-Attenuated Ex

Smallpox, oral poliovirus, measles

Killed-Inactivated Vaccine

Inactivated pathogen
By heat, chemicals, radiation
No replication

Killed-Inactivated Advantages

Safer: No mutation/replication
Easy storage and transport

Killed-Inactivated Disadvantages

Require boosters
Injected

Killed-Inactivated Ex

Rabies, influenza

Toxoid Vaccine

Inactivated toxin
Pathogen product

Toxoid Advantages

Safe: No replication/spread
Stable: No reaction to temp, humidity, light

Toxoid Disadvantages

Require boosters
Require adjuvant (substance enhancing immune response)

Toxoid Ex

Tetanus, diphtheria

Subunit Vaccine

Pathogen fragment inducing immune response

Subunit Advantages

Safest

Subunit Disadvantages

No long-term immunity
Require boosters
May require carrier (attach stronger antigen)

Subunit Ex

Hep B

mRNA Vaccine

Produce viral proteins
Recruit immune cells
APC present proteins
Induce B-cell (antibodies) and T-cell immunity

mRNA Ex

SARS-CoV-2

mRNA Vaccine 1: Vaccine Production

Produce mRNA encoding antigen protein
From viral DNA template
Add to vaccine formula

mRNA Vaccine 2: Host Cell

Lipid-enveloped mRNA enter host cell
Produce protein

mRNA Vaccine 3: APC

Protein exit cell
APC internalize and process protein into peptide/antigen
APC display antigen on surface via MHC

mRNA Vaccine 4: Immune Response

Helper T-cell recognize antigen
Initiate immune response
B-cells produce antibodies
T-cells destroy infected cells

Antiviral Medications

Treat COVID
No infection prevention

Antiviral: Molnupiravir

Polymerase inhibitor (replication and transcription)
Increase viral RNA mutations
Impair viral replication

Antiviral: Nirmatrelvir

Protease inhibitor (cut protein into functional pieces)
Inhibit viral protein assembly and replication
Ritonavir protection

Herd Immunity

Indirect protection from infectious disease
Large population immunity slows/stops disease spread

Herd Immunity: Reproduction Number (R0)

Measure herd immunity
Determine threshold immunization coverage to eliminate disease
Increase R0 = increase immunization coverage

Vaccine Development 1: Lab Studies

Research using assays
Identify infectious agent epidemiology
Select strain/subtype for vaccine
Develop and test manufacturing process

Vaccine Development 2: Preclinical Studies

Research using animal models
Evaluate immunogenicity and toxicity
Pharmacological effects and safety

Vaccine Development 3: Clinical Phase 1

Small-scale human trials (10-100)
Assess local and systemic reactions (safety)
Determine immunogenicity and immune response production

Vaccine Development 4: Clinical Phase 2

Larger scale trials (50-500)
Assess safety, side-effects, efficacy
Determine optimal dose and schedule

Vaccine Development 5: Clinical Phase 3

Large scale trials (300-30,000)
Assess efficacy and safety under natural disease conditions
Apply for licensing

Vaccine Development 6: Health Canada Approval

Regulatory authority ensures quality, safety, and efficacy
Evidence for approval

Development Challenge: Cost

Premature research abandonment

Development Challenge: Cold Chain

Live-attenuated vaccines must be kept cold
Broken cold chain = vaccine lose potency/efficacy

Development Challenge: Continuous Monitoring

Monitor evolving pathogens changing/losing antigenic determinants
Vaccine lose efficacy

Development Challenge: Gold Standard

"Best" vaccine new vaccines must surpass
Ex: Recombinant vector, improve vaccine adjuvant

Development Challenge: Influenza Virus

Virus structure prevent one-for-all vaccine

Influenza: Neuroaminidase Antigen

Surface protein remove sialic acid from cell surfaces
New viral copies infect and spread
11 subtypes

Influenza: Haemagglutinin Antigen

Surface protein bind sialic acid on glycoproteins
Promote endocytosis
Fuse endosome and viral membrane
18 subtypes

Cancer

Error in cell growth and death balance mechanisms
Uncontrolled cell growth

Cancer Cells

Altered self cells escaping normal growth-regulating mechanisms
No growth factor for division
No response to signals stopping division
DNA changes induce cell transformation and malignancy
Resilient against stressful environment
Causes: Carcinogenic chemicals, radiation

HeLa Cells

Immortal ovarian cancer cells
Allow for research breakthroughs

Tumour

Abnormal tissue mass of cancerous cells

Benign Tumour

Non-cancerous
Cannot grow indefinitely and invade tissues

Malignant Tumour

Cancerous
Grow indefinitely and invade tissues
Metasize: Colonize other sites by travelling through blood/lymphatic vessels

Hot Tumour

T-cell inflamed
High immune activity (CD8+ TIL, interferon gene)
Responsive to treatment

Cold Tumour

T-cell non-inflamed
Low immune activity (CD8+ TIL, interferon gene)
Less responsive to treatment

Cancer-Immunity 1: Cancer Antigen Release

Cancer cell death release antigens
Recognized by immune system

Cancer-Immunity 2: Antigen Presentation

APCs and dendritic cells capture antigens
Travel to lymph nodes for T-cells

Cancer-Immunity 3: Priming and Activation

APC antigen presentation activate T-cells
Initiate immune response

Cancer-Immunity 4: T-Cell Trafficking

Activated cytoplasmic T lymphocytes (CTL) move through blood vessels to tumour

Cancer-Immunity 5: T-Cell Infiltration

CTL pass through endothelial cells
Invade and attack tumour cancer cells

Cancer-Immunity 6: Cancer Cell Recognition

CTL recognize cancer antigen

Cancer-Immunity 7: Killing Cancer Cells

T-cells and immune cells initiate pathway to kill cancer cells

Immunosurveillance

Healthy immune system identify and control tumour cells
Failure = Cancer cells evade recognition
Anti-tumour response does not kill all cancer cells

Immunoediting

Process connecting tumour cells and immune system
Immune system destroying cancer cells promotes tumour growth

Immunoediting 1: Elimination

Immune cells recognize and eliminate tumour cells
NK cells, cytotoxic and helper T-cells

Immunoediting 2: Equilibrium

Non-eliminated cells enter equilibrium
Cell proliferation = cell death

Immunoediting 3: Escape

Immune system does not recognize tumour cells
Avoid elimination
Uncontrolled cancer cell proliferation forms tumours

Immune Response Evasion: Reduce MHC Expression

Low MHC class 1 on tumour cells
CTL cannot recognize

Immune Response Evasion: Poor Costimulatory Molecules

No costimulatory molecules in tumour cells
CTL partially activated

Traditional Cancer Treatment

External drugs selectively target and kill cancer cells

Traditional Cancer Treatment: Interferons

Stimulate activity
Convert cold tumours to hot

Traditional Cancer Treatment: Immune Checkpoint Inhibitors

Prevent immune response regulation
Continue attacking tumour

Immunotherapy

Mark cancer cells for immune system to eliminate

Immunotherapy Advantages

Attack cancer types in all organs
Specific cancer cell elimination (protect healthy cells)
Develop immunological memory
Enhance anti-tumour immune responses (cancer antigen presentation, cancer cell elimination)

Tumour Infiltrating Lymphocytes (TIL)

Immune cells as prognostic biomarkers
T-cells, B-cells, NK cells, dendritic cells, macrophages
Evaluate disease course and therapeutic intervention response

TIL: Chemotactic Gradient

Induce TIL migration to tumour
Formed by chemokines

TIL: Phenotypic Markers

Reflect TIL activation status
Impact biomarker role
TIL amount not the same as TIL activity

Immunoscore

Measure T-cell density in tumour center (CT) and periphery (IM)
Separate low and high risk cancers
Develop treatment plans

Immunoscore 1

Separate tumour in CT and IM regions

Immunoscore 2

Stain T-cells and conduct digital pathology

Immunoscore 3

Assign tumour score relating to diagnosis/risk level