Labeling techniques for immunofluorescence microscopy and flow

cytometry

Kuby, Kindt, Goldsby, Osborne Immunology Textbook

Flow cytometry --Fluorescence Activated

Cell Sorting (FACS)

A two-color FACS analysis

Kuby, Kindt, Goldsby, Osborne Immunology Textbook

Single color FACS analysis (e.g., using anti-A antibody) -- note this is a log scale

A- cells

A+ cells

Stanford used flow cytometry to screen blood before HIV tests were available

• Reduced ratio of CD4+ to CD8+ T cells in AIDS patients

• July 1983 to June 1985, Stanford Blood Center used flow cytometry to test donated units for CD4:CD8 ratio

• Did not transfuse blood from donors with CD4:CD8 ratio < 0.85

• Most other blood banks did no screening

• ~10,000 cases of transfusion-transmitted AIDS in US before HIV test available in 1985

Galel et al., 1995, “Prevention of AIDS transmission through screening of the blood supply” Annu. Rev. Immunol. 201-227

Clicker question

Swine flu is a dangerous form of influenza. Which of the following is true:

1) Swine flu is highly contagious amongst pigs, but does not infect humans.

2) Swine flu is normally spread only amongst pigs, but can be caught by humans by eating infected pork products.

3) Swine flu can be caught by humans through contact with infected pigs and/or their environment. However, there are NO documented cases of human-to-human spread.

4) Swine flu can be caught by humans through contact with infected pigs and/or their environment. Many of the current swine flu cases resulted from human-to-human spread.

Clicker question

Bird flu is a dangerous form of influenza with a high fatality rate. Which of the following is true:

1) Bird flu was created accidentally by making flu vaccines in hen eggs.

2) Bird flu is highly infectious in birds, but does not infect humans.

3) Bird flu can infect humans and birds. Humans are infected only through direct contact with infected birds (no human-to-human transmission).

4) Bird flu can infect humans and birds, but there are very few documented cases of human-to-human transmission. Only a few mutations could convert it so that it is easily transmitted between humans.

5) Bird flu can infect humans and birds. There are currently many documented cases of human-to-human transmission. A vaccine against bird flu is already available and is responsible for reducing the number of human-to-human transmissions of bird flu.

Swine flu has pandemic potential

• New strain of swine flu is H1N1, related to 1918 pandemic strain

• Spreads efficiently between humans– Killed >100 people in Mexico– Infected >100 people in the US in 37 states. 1 death.– Been found in countries outside of North America– (Statistics for early May 2009)

• Novel variant -- similar to H1N1 swine flu strains that infect only pigs, but this H1N1 shows human-to-human transmission. Does not require contact with pigs to be infected.

http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm

http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm

Influenza (flu)

• A respiratory virus acquired by inhaling– Usually transmitted by coughing, sneezing

• Virus-containing drops expelled at 100 miles/hour• Each drop has millions of viruses• Need ~10 viruses to get infected

– Viruses can survive for several days on telephone, door knobs, etc., but are disrupted by detergents

• Cytolytic virus -- kills the cells it infects

• Induces the host to produce interferon, which causes flu symptoms

•Negative strand RNA virus– Influenza A has 8 negative single strands

Clicker question

Flu viruses must carry what type of enzyme into infected host cells in order to produce new viruses?

1) Reverse transcriptase2) RNA-dependent DNA polymerase3) RNA-dependent RNA polymerase4) Integrase5) Restriction enzyme6) None -- RNA can be translated directly to

make new viral proteins

Influenza virus

• Enveloped virus

• Three surface proteins

• Hemagglutinin (H)• Neuraminidase (N)• M2 ion channel

• Nucleocapsid protein

• 8 Negative strand RNAs

Influenza A• Flu (Influenza A) is caused by viruses that undergo

continuous antigenic change and possess an animal reservoir. (Influenza B & C only infect humans.)– Antigenic changes affect recognition by the host immune system.

• Two major glycoproteins on surface: hemagglutinin (H) and neuraminidase (N). – At least 15 different hemagglutinin subtypes – At least 9 different neuraminidase subtypes

• Human influenza viruses generally limited to H1, H2, H3 and N1 and N2 subtypes. Birds are hosts for other subtype strains.

• Changes in surface glycoproteins have caused epidemics and pandemics* in humans -- 1918 flu pandemic was caused by an H1N1 virus.*Pandemic: When an epidemic spreads throughout the world

Influenza pandemics

Due to Spanish Flu

M1 Matrix protein

Hemagglutinin

Neuraminidase

M2 ion channel

Viral ribonucleoprotein

Nucleoprotein

Viral RNA

1. Virion enters cell by receptor-mediated endocytosis. Taken into endosomes.2. Low pH allows fusion. M2 ion channel makes inside of virus more acidic. M1 dissociates and viral ribonucleoproteins enter nucleus.3. Viral polymerases transcribe (3a) and replicate (3b) the RNA.4. Newly-synthesized mRNAs go to cytoplasm, where they are translated.5. Post-translational processing of viral proteins in Golgi (5b). Nucleoprotein goes to nucleus (5a).6. Nucleoprotein binds new RNAs, go to region of cell surface with H, N and M2.7. Budding.

http://www.ncbi.nlm.nih.gov/genomes/FLU

Infection by influenza virusHost cell receptors(sialic acid-containing glycoproteins)

Flu viruses get taken into acidic endosomes by receptor-mediated endocytosis. Low pH induces fusion of the flu virus membrane with the endosomal membrane, resulting in release of viral RNA into the cytoplasm.

HIV fuses at the surface of the host cell -- doesn’t require low pH for fusion.

pH 7.4

pH ≤6.0

Influenza virus hemagglutinin (H or HA) binds to sialic acids on host cell glycoproteins

• Flu virus H is a trimer. Each subunit contains two chains: HA1 and HA2.

• HA1 (gray) contains the sialic acid binding site. HA1 is the counterpart of HIV gp120.

• HA2 (colored) is responsible for fusion. HA2 is the counterpart of HIV gp41.

• Sialic acid is the host cell receptor (counterpart of CD4).

Sialic acid

Sialic acids are components of complex carbohydrates on cell surface glycoproteins.

Different hemagglutinins are specific for different isoforms of sialic acid,

hence defining host range of flu viruses.

Original H structure: Wilson et al., 1981, Nature 289: 366-373

Hemagglutinin trimer

Nature Structural & Molecular Biology 16, 233 - 234 (2009)

Antibodies bind around the receptor binding pocket, but can’t access it completely (Canyon Hypothesis).

Hemagglutinin (H) undergoes a large structural rearrangement when exposed to low pH

H monomer at neutral pH: fusion inactive

H monomer at acidic pH: fusion

active

Receptor binding site

Fusion peptide

Fusion peptide

Viral membraneViral membrane

Acidic pH: fusion active

Note that predicted position of viral membrane has

changed.

Neutral pH: fusion inactive

Fusion peptide

Clicker question

Residues in the hemagglutinin and HIV fusion peptides are primarily

1) Hydrophobic2) Hydrophilic

Carr and Kim, 1993, Cell 73: 823-832

A spring-loaded mechanism for the conformational change of influenza

hemagglutinin

HA1HA1

fusion peptide

fusion peptide

Viral membrane

HA2HA2

Note that hemagglutinin, like gp120/gp41, is actually a trimer -- what is shown schematically is the structure of a monomer.

Analogy to fusion by HIV:HA1 is the counterpart of gp120.

HA2 is the counterpart of gp41.

The trigger for hemagglutinin for changing to its fusion-active form is low pH. The trigger for HIV spike protein for changing to its fusion-active form is binding to CD4 and CCR5.

Conformational changes also alter position of viral membrane -- not shown

here.

Membrane fusion

Fusion Mechanism

Clicker questionInfluenza is an enveloped virus that buds out from the host cell membrane, incorporating flu proteins, including hemagglutinin, into its envelope. Influenza hemagglutinin binds to sialic acid, which is a component of N-linked carbohydrates attached by host cell glycosylation machinery. How does flu virus manage to bud from a cell that includes lots of sialic acid-containing proteins at its cell surface? (Wouldn’t it get stuck?)

1) Flu virus encodes an enzyme that cleaves sialic acids from host and viral proteins.

2) Once inside an infected cell, flu virus shuts down host cell machinery for attaching sialic acids.

3) Newly-budded flu viruses remain attached to host cells via interactions between hemagglutinin and sialic acid-containing host cell proteins.

4) Fevers that accompany influenza infection promote release of newly-budded flu viruses from host cells.

Neuraminidase monomer with bound inhibitor

Influenza neuraminidase is an enzyme that cleaves sialic acids so that budding influenza viruses can be released from host

cells

Original neuraminidase structure: Varghese et al., 1983, Nature 303: 35-40

Palese, 2004, Nature Medicine 10: S82-S87

Inhibition of influenza replication cycle by antivirals

• Two classes of FDA-approved drugs available against influenza A:

• M2 blockers (e.g., amantadine, rimantadine) prevent influx of protons through M2 ion channel, preventing release of viral genes into cytoplasm.

• Neuraminidase inhibitors (e.g., oseltamivir (Tamiflu), zanamivir) prevent release of virus from infected cell.

• Drug resistance has been reported for both antiviral classes.

Swine flu (H1N1) is resistant to amantadine and rimantadine (M2 blockers)

Swine flu is sensitive to oseltamivir (Tamiflu) and zanamivir (Neuraminidase inhibitors)

Figure 11-2

Antigenic drift

• Results from error-prone copying of viral RNA.

• Responsible for local outbreaks of influenza infections that occur every year or so.

• Antigenic drift explains why we get the flu more than once.

• Usually point mutations, so new variants cause only mild disease in previously-infected people (because some of their antibodies are still effective).

Hemagglutinin (H) is the viral surface protein responsible for entry into host

cells.

Artist’s rendition of antigenic

shift

http://pr.caltech.edu/periodicals/CaltechNews/articles/v41/uneasypieces.htmlLicensed from http://www.rkm.com.au/

• Hybrid viruses produced in a secondary host -- result from reassortment of segmented RNA genomes

• Antigenic shift is rare, but shifted strains cause the flu pandemics that occur every 10-50 years.

• What is the “secondary host”?

Antigenic shift

Antigenic shift in Type A influenza

Avian versions of influenza A cause digestive tract infection in birds.

Pig can be host to both avian and human influenza A. Some hybrid viruses can infect humans.

Reassortment of viral gene segments most likely in places where pigs, ducks, and humans live in close contact.

Sequences of 2009 H1N1 Flu

http://www.ncbi.nlm.nih.gov/genomes/FLUAlso includes links about flu virus biology

Early reports said 2009 swine flu was mixture of human, bird and pig influenzas.

Later reports said 2009 swine flu is a reassortment from two pig flu viruses (North American and Eurasian swine flu viruses).

The N1 gene is from Eurasian swine flu -- never before seen in humans.

Flu vaccines• WHO* specifies the contents of the vaccine

each year to contain the most likely strains of viruses that will attack the next year.

• Annually updated trivalent flu vaccine for 2008-2009 season consisted of H3N2, H1N1 and B influenza viruses. (But the vaccine H1N1 strain doesn’t confer protection to H1N1 swine flu.)

• Viruses are grown in hens’ eggs.

• Inactivated viruses are injected or live attenuated viruses are given as a nasal spray (not currently approved in children younger than 5).

*World Health Organization

H5N1 (Bird flu)• Most flu viruses infect birds. “Bird flu” refers to influenza in birds

and in humans in cases in which avian viruses cross the species barrier to infect humans.

• 2003-2004: Outbreaks of H5N1 in poultry in 8 Asian countries and 100 cases of human infections with 50% fatalities.

• Few (maybe none) cases involved bird-to-human transmission. A pandemic would require human-to-human transmission. A change of only two amino acids would allow H5 to efficiently recognize human cells.

• H5N1 viruses are being tested for making a vaccine, but mass production of an H5N1 vaccine would require safety and efficacy trials that might take longer than viruses would spread in a pandemic.

• Antiviral drugs oseltamivir (Tamiflu) and zanamivir, but not amantadine, are active against H5N1. Supply not adequate for a global pandemic. Antiviral resistance occurs in N1 viruses during Tamiflu treatment.

Response to bird flu threat in Asia

Protect domestic ducks Burn chicken carcasses Vaccinate chickens from contact with wild ducks

http://pr.caltech.edu/periodicals/CaltechNews/articles/v41/uneasypieces.html