Immunology Lim 5-10

What does vagina release to support commensals

Glycogen, which feeds the good bacteria

3 ways in which bacterial pathogens harm us

1. Release of endotoxins (such as tetanus)

2. Colonization of epithelium surface (streptococcus)

3. Tissue damage by colonizing in bulk or replicating intracellularly (like leprosy)

4. Most use something in between the above methods (Like Staphylococcus aureus)

List modes of actions of exotoxins

1. Pore formation

2. Enzymatic lysis

3. Inhibition of protein synthesis

4. Hyperactivation

5. Effect on nerve-muscle junctions

How does pore formation work when used by exotoxin

1. Released by Staph. aureus

2. Polymerizes on cell surface

3. Cell nutrients leak

How do exotoxins perform cell lysis

1. Alpha toxin (Phospholipase C) released by C. perfringens

2. Hydrolyzes phosphorylcholine causing cell lysis

How do exotoxins inhibit protein synthesis

1. Diphtheriae toxin enters cell

2. Blocks activity of elongation factor 2

3. Protein synthesis is blocked

4. Cell dies

How does exotoxin cause hyperactivation of cells

1. Cholera bacteria releases toxins

2. Toxin causes hyperactivation of adenylate cyclase

3. A lot of cAMP is made

4. Excess nutrients and electrolytes are released, causing diarrhea

How do exotoxins affect the nerves

Inhibitory, Botulism

1. Botulinum toxin stops release of acetylcholine

2. Muscles cannot be stimulated

Activator, tetanus

1. Tetanus toxin stops release of inhibitory signal

2. Constant neuron stimulating signals are sent

Typical structure and function of exotoxin

Two chain molecule, one chain is for entering the cell, the other chain is for performing toxic function

4 ways in which immune system kills bacteria

1. Complement mediated lysis

2. Chemotaxis

3. Phagocytosis

4. Killing using proteolytic enzymes or free radicals

What chemotaxis is found on microorganisms which facilitates immune system killing of them

FMLP-peptide on the N-terminal

5 ways in which phagocytosis can occur

1. Complement receptors binding to bound complements

2. Fc receptors binding to antibodies

3. PRRs recognizing PAMPs

4. LPS receptor binding to LPS

   1. Receptors are CD18 and CD14

5. Lectins (Like macrophage mannose binding lectin)

Result of CD14 binding to LPS

Phagocytosis and TNF secretion by macrophage

Which cells express TLRs

1. Macrophage

2. Neutrophil

3. Dendritic cell

TLR5 detects what

Flagellin

TLR1/2 detects what

Triacyl lipopeptides

TLR3 detects what

dsDNA

TLR2/6 detects what

Diacyl lipopeptides

TLR2 detects what

Peptidoglycan lipoarabinomannan

TLRs share a common intracellular pathway which leads to

1. Secretion of proinflammatory cytokines by macrophages (TNFs, IL-1)

2. ROS production by neutrophils

3. Upregulation of costimulatory molecules on APCs

TLRs intracellular pathway (Lim version)

How do oxygen metabolites participate in phagocytosis

1. Bacteria is in phagosome

2. NADPH oxidase pumps O2 into the phagosome

   1. Creates ROCs like O2 → OH radical, H2O2, and O-

3. This is toxic to bacteria

4. If lysosomes fuse, they add myeloperoxidase, and peroxisomes add catalase

5. These, in presence of halides electron donors, create more toxic oxygen species like hypohalites

   1. HIO

   2. HClO

Nitric oxide pathway

Made is response to IFN-gamma signaling

1. Inducible nitric oxide synthetase is made as a result

2. i-NOS catalyzes a reaction between oxygen and guanidine side chain or arginine to yield nitric oxide

   1. These can form peroxynitrites with ROCs

Made in response to TH2 signaling, IL-4/10/13

1. Arginase converts Arginine into urea and ornithine

2. Polyamines can be used for collagen synthesis and cell proliferation

Lysozyme role

Destroy proteoglycan

6 ways how microbes can fight off complement system

1. Send decoys which inhibit binding of complements

2. Made a capsule to block binding of complements

3. Express enzymes on surface to destroy complements

4. Express proteins to disrupt complement receptor binding to complements

5. Express proteins to divert complement binding

6. Membrane can resist insertions of lytic complex

Evasion mechanisms bacteria use to fight off antibodies

1. Release enzymes to cleave antibodies

2. Express fc-like receptors to stop opsonization

Roles of antibodies when fighting bacteria

1. Neutralize toxins

2. Complement activation

3. Opsonization

4. Neutralize immunorepellents

5. Block transport mechanisms and receptors of bacteria

6. Neutralize factors and enzymes released by bacteria

Evasion mechanisms of bacteria to escape phagocyte mediated killing (9+1)

1. Secrete toxins to kill phagocyte

2. Secrete toxins to neutralize chemotaxis

3. Special capsule to prevent complement formation and phagocytosis

4. Secrete molecules to prevent lysosome-phagosome fusion

5. Secrete catalase to destroy free radicals like H2O2

6. Special capsule to resist free radical damage

7. Stop antigen presentation

8. Escape phagosomes and multiply in cytoplasm

9. Prevent IFN-gamma responsiveness by macrophages

10. Inhibit H+ pump in phagosome to stop free radical damage

Three things that happen to tissue upon inflammation

1. Increased blood flow

2. Increased transduction of molecules into the tissue

3. Increased presence of white blood cells

Which complements trigger mast cells to release their granules

C3a and C5a

list 5 mediators of inflammatory response

1. C3a

2. C5a

3. Histamine

4. IL-8

5. PAF (Platelet activating factor)

2 ways virus spreads between cells

1. Release of virions

2. Cell to cell contact

Two ways to fight a virus infection

1. Prevent

   1. Use interferons (innate immune system)

   2. Antibodies to neutralize then (adaptive)

2. Kill virus

   1. Kill infected cells via ADCC by NK cells

   2. Induce apoptosis, by T cells

   3. NK cell cytotoxicity

First line of defense against viruses

IgA and interferons

Two main strategies, and their sub strategies, which viruses use to survive in nature

1. Change of coating

   1. Antigenic drift (mutation)

   2. Antigenic shift (2 strains combine to form a new virus)

2. Latency

Sources of different interferons

1. Alpha and beta - made by all cells

2. Gamma - T cells and NK cells

Which interferon regulates macrophage action

Gamma

Effect of interferons on MHC upregulation

1. Alpha and beta upregulate MHC I

2. Gamma upregulates MHC I and II

What happens upon IFN signaling which aids in preventing the spread of viral infection

1. Synthesis of 2’5’ oligoadenylate synthetase

   1. Degrades viral mRNA

2. PKR is synthesized, inhibiting elF-2 by phosphorylating it

   1. Stops protein synthesis

3. Mx protein is synthesized and phosphorylated causing it to polymerize

   1. Inhibits virus transcription and assembly

Gamma interferon activates which cells

1. Macrophage

2. NK cell

Effects of interferon gamma

1. Upregulation of MHC I and II

2. Activation of macrophages and NK cells

3. Blocking virus replication

Two types of parasites

1. Protozoa (unicellular)

2. Worms (multicellular)

How to fight two types of protozoans

Intracellular

1. Antibodies are ineffective

2. Cellular mechanisms are involved

Extracellular

1. Antibodies allow for ADCC, complement fixation and phagocytosis

How to fight worm infections

1. Antibodies mediate complement binding

2. Antibodies also mediate toxic granule release by eosinophils and IgE dependent mechanisms

How would macrophages fight intracellular pathogen

Making reactive oxygen and nitrogen species

How to boost the ability of macrophages to fight intracellular pathogens

Release cytokines which can increase the production of toxic metabolites

Roles of antibodies in fighting parasites

1. Direct damage via complement fixation

2. Enhance opsonization

3. Block the spread of pathogen by blocking the receptor they need to enter cells, such as plasmodium

4. ADCC

Which cells recruit eosinophils cells

Mast cells release mediators which recruit eosinophils

What do eosinophils use to attack worms

1. Release Major basic protein (MBP)

2. Reactive oxygen species

Factors needed to produce eosinophils (3)

All come from T cells

1. IL-5

2. Eosinophil stimulating promoter (ESP)

3. IL-4 → Bone marrow stem proliferation

Result is production of eosinophils

Mechanisms of eosinophils activation full diagram

List where HIV can be isolated from

1. Lymphocytes

2. Urine

3. Blood

4. Saliva

5. Semen

6. Female genital tract secretion

7. Tears

8. Breast milk

Modes of transmission of HIV

1. Sexual

2. Organ donation

3. Needle sharing

4. Mother to child

Three waves of aids transmission in the US

1. Homosexual

2. Drug abusers

3. Heterosexual

Describe structure of HIV virion

1. envelope glycoprotein

   1. gp120 and gp41

2. Envelope derived from host cell

3. Matrix protein p17

4. Structural core protein p24, p17, p9, p7

5. Reverse transcriptase

6. ssRNA

List genes of in HIV genome and purpose

1. LTR - for genome integration

2. gag - virion core proteins and antigens

3. pol - reverse transcriptase and integrase

4. vif - virion infectivity factor

5. vpr - TF

6. nef - negative activator factor

7. tat - transactivator protein

8. ref - regulatory virion protein

9. vpu - efficient budding

10. env - envelope proteins

Two steps of how HIV infects cell

1. gp120 binds to CD4 (Also CCR5 and CXC4)

   1. Found on helper T-cells, macrophages, monocytes, and FDCs

2. gp41 helps the virion fuse with the host cell membrane

3. T-cells usually get killed by viral replication, but other cells will continue to carry the virus

Stages of a typical HIV infection

1. Initial infection, symptomatic

2. Seeding of virus across lymphoid organs

3. Latency period

   1. Asymptomatic

   2. T-cell count becomes lower

4. Constitutional symptoms (plasma viremia starts increasing)

5. Opportunistic infections

6. Death

Some opportunistic infections and tumors indicative of AIDS

1. Tumor - Kaposi’s sarcoma

2. Bacteria - mycobacteria and salmonella

3. Fungi - Pneumocytes carinii

Disease stages of AIDS

1. Contact

2. Seroconversion - detection of HIV antibodies

3. Sickness

   1. Stage 1 - Acute

   2. Stage 2 - Asymptomatic

   3. Stage 3 - persistent generalized lymphadenopathy (PGL) - enlarged lymph nodes

   4. Stage 4 - Symptoms, persistent infections, neurological problems, tumors

Laboratory markers of stage IV AIDS

1. Decreased CD4+ T cells

2. Increased IgE, IgA and Beta2 microglobulins

3. Reduced anti-gp120, anti-p17 and anti-p24 antibodies

4. Raised CD8+ T cells

3 classes of anti-HIV drugs

1. Nucleoside analogs like AZT which block transcription

2. Protease inhibitors stop assembly of viral particles

3. Non-nucleoside reverse transcriptase inhibitors

How can HIV develop resistance to anti-HIV drugs? How to fix?

1. Mutations

2. Use cocktail treatment

2 PrEP treatments

1. Truvada - for safety before intercourse and drug use

   1. Cytidine analog and TDF

   2. Both are RT inhibitors

2. Descovy - idk what he tried to say

   1. Cytidine analogue and TAF

   2. Both are RT inhibitors

TAF vs TDF as RT inhibitors

TAF is a newer version, absorbed faster

What to look for when diagnosing HIV?

Antibodies

Why not detect viral antigens when testing for HIV?

Because titre of virus is low in blood

What test is used to check for HIV antibodies? Basic steps

ELISA

1. Cover glass with HIV antigens

2. Add patient serum

3. Add anti-human IgGs with enzyme conjugated

4. Add ligand

5. If antigen is present, color will change

Confirmatory tests after ELISA

1. PCR

2. Western Blot

Difficulties in making HIV vaccine

1. Antigenic drift of gp120

2. Antibodies fail to protect the patient

3. Possibility of cell-cell transmission

Logic behind vaccines

1. Introduce antigens to activate T-cell response

2. T-cells form memory cells

3. They will activate faster when the infection occurs again

What does specificity mean in immunity

TCRs and BCRs are unique

An ideal vaccine should generate both

1. Humoral response (by B cells)

2. Innate response (by T cells, cytotoxic T cells)

5 ways to make vaccines

1. Attenuated live vaccine

   1. Animal virus (like cowpox)

   2. Non-virulent strain

   3. Serially passage the virus to accumulate mutations till they lose virulence

2. Killed pathogen vaccine

3. Subunit (recombinant protein) vaccine

4. mRNA/DNA vaccine

5. Recombinant virus vaccine

How is Recombivax HB vaccine produced

Grow viral subunits is yeast cells

How is Engerix B vaccine made

How is polio inactivated

Using beta-propiolactone

Some methods of inactivating bacteria

Heat and formaldehyde

How do recombinant live virus vaccines work

1. Get vaccinia virus

2. Insert a gene of interests coding for antigen of a specific microorganism

3. Insert it into cells which will make virions

4. Collect virions for a vaccine

5. Inject into cells

6. Vaccinia proliferates and causes production of antigen

7. Body develops immunity against the antigen

How do conjugation vaccines work

1. Link a weak and strong antigen together

2. Body will react to strong antigen, and also remember the weaker antigen

3. Stronger antigen is called carrier protein

How does DNA vaccine work

1. Inject DNA

2. DNA codes for antigen

3. Antigen gets recognized and memorized

How mRNA vaccines work

1. mRNA uses lipids to enter the cell

2. Transcribed as antigen

3. Antigen gets presented on MHC I

4. If it enters another cell, it is presented on MHC II

Why not used killed virus vaccines?

1. Some organisms cannot be grown to sufficient quantity

   1. Hep. B

2. Some won’t trigger an immune response

   1. No mucosal immunity, i.e. pathogen can enter the body again

Example vaccine which shows why live attenuated may be better than dead vaccines

1. Polio vaccine is live attenuated virus

2. Orally administered

3. Triggers production of nasal and duodenal IgA, while dead vaccine does not

4. Therefore better immunity

Why is cholera vaccination not as effective as typhoid vaccination?

1. Cholera resides in the gut

2. Typhoid enters organs

3. Vaccination causes production of circulating antibodies, which cannot reach the gut but can reach all organs

6 important criteria for a good vaccine

1. Safe

2. Effective long-term

3. Cost effective

4. Memory immunity

5. Duration of response

6. Stable (can be transferred)

Table comparing inactivated, live and mRNA vaccines in following categories. Preparation, administration, Adjuvant, safety, heat liability, cost, duration of immunity, immune response

What characteristic of viral proteins causes antigenic shift

DNA/RNA polymerases lack proofreading ability

Serotype

A way of grouping pathogens according to whether they illicit an immune response. H1 influenza infection will not create antibodies for H2 influenza

Influenza virus has what two molecules on surface

1. Hemagglutinin (H)

2. Neuraminidase (N)

Formula for vaccine effectiveness

VE=100*(1-IRR)

where IRR=( Trial / Control )

mRNA vaccine delivery method

Lipid nanoparticles

What does mRNA vaccine LNP contain

1. uRNA

2. saRNA

3. modRNA

How do anti-S antigen antibodies work to stop COVID19 infection

They bind the spike protein, preventing the virus from infecting cells

Steps in production of inactivated vaccine

1. Produce virus using cells

2. Inactivate virus

3. Validate inactivation

4. Purification

   1. Size exclusion chromatography

   2. Filtration

Why is omicron a dangerous mutant

Because it can more effectively escape neutralization of antibodies produced in response to previous vaccines

IFN-alpha/Beta vs IFN-gamma in terms of Antiviral activity, and which cells secrete them,

1. IFN-alpha/beta are antivirals, IFN-gamma is pro-inflammatory

2. T cells produce IFN-gamma primarily, while all infected cells produce IFN-alpha/beta

Stems of IFN release during viral infection

1. Cell is infected, detects it with TLRs and releases type I interferons

2. Virus gets picked up by type I IFN-stimulated APCs, and its antigen is presented to T cells

3. T-cells release IFN-gamma to trigger macrophage and NK cell differentiation, as well as Th1 mediated response

Where can virus replication take place in infected cells

1. Cytoplasm

2. Nucleus

How fast do viruses replicate

Fucking fast