The branch that breaksIs called rotten, butWasn’t there snow on it?

Bartolt Brecht

Haiti after a hurricane

Your body has evolved complexmechanisms of recognizing “non-

self”and fighting against it

The Immune System is the Third Line of Defense Against

Infection

Antibodies are Produced by B Lymphocytes(B cells totheir friends)

T Lymphocytes(T cells)provide“cell based”immunity

Overview of Human Immunity:Adaptive and Innate

Lymphocyte Origins

16-22

Let’s start with the role of B cells and antibodies in the immune response

Some definitions are in order

Antigen

A substance produced by a pathogen (e.g., protein, complex sugar) capable of producing an immune response

Some definitions are in order

Antibodies

Protein molecules (immunoglobulins) produced by B lymphocytes to help eliminate an antigen

Molecular Biology of the Cell Alberts et al

B cells Make

AntibodiesIn response to

antigens

Molecular Biology of the Cell Alberts et al

These antibodiescan bind to

and “neutralize”Viruses

or can directimmune attack

of virus-infectedcells

Molecular Biology of the Cell Alberts et al

Antibodies can also direct phagocytosis of pathogens

Cytotoxic (Killer) T Cells Recognize, Attack and Kill

Virus-Infected Cells

CELLS alive!

Let’s focus first on antibodies

Molecular Biology of the Cell Alberts et al

Molecular Biology of the Cell Alberts et al

Antibodies are proteins that have evolved to recognize molecules from pathogens

These molecules from pathogens are called Antigens

Molecular Biology of the Cell Alberts et al

The variable and constant regions ofantibodies are related = Ig domains

Constant Region

Hypervariable Region

Light Chain

Heavy Chain

Antigen Binding Region

Let’s use as an example an antibody that recognizesa protein on the surface of flu (influenza) virus

courses.washington.edu/medch401/pdf_text/401_07_lect2.ppt

Hemagglutinin

Here is the antibodyBound to the “antigen” = influenza hemagglutinin

Human antibody

Rotate ~90

Add all atoms

The antibody recognizes the antigen by a lock-and-key fit

Antigen residues at the interface = epitope

Epitopes are typically ~5 residues long

This interaction is VERY specific

hemagglutinin

antibody

Space-filling mode

Grey now = mainchainof hemagglutinin

Epitopes reside in turns and loops

This interaction is VERY specific

You can generate antibodies against HIV

like you do against other viruses

Molecular Biology of the Cell Alberts et al

Given thousands of pathogens each of which is constantly evolving

how do we generate antibodies against each?

Molecular Biology of the Cell Alberts et al

We cannot dedicate all 25,000 genes in the genome

just to make antibodies.

What’s the solution?

Molecular Biology of the Cell Alberts et al

We cannot dedicate all 25,000 genes in the genome

just to make antibodies.

What’s the solution?

Put antibodies together by a mix-and match approach!

Molecular Biology of the Cell Alberts et al

requires rearranging the DNA

Molecular Biology of the Cell Alberts et al

requires rearranging the DNA

Molecular Biology of the Cell Alberts et al

The result:an antibody light chain

Since there are multiple types of each gene segment, there are

thousands of possible V-D-J combinations

Each B cell gets a unique combination

Since there are multiple types of each gene segment, there are

thousands of possible V-D-J combinations

Each B cell gets a unique combination

Other mechanisms

further increaseantibodydiversity

Molecular Biology of the Cell Alberts et al

When a pathogen enters the bodyit stimulates proliferation of the

specific B Cells that recognize its Antigens

Once you are exposed to an antigenyour B cells “remember” this

CELLS alive!

OK, that explains antibodies and B

cellsbut what about us?

Molecular Biology of the Cell Alberts et al

T cells carry antibody-related proteinson their plasma membranes

called T cell receptors

Molecular Biology of the Cell Alberts et al

T cell receptors are also assembledby gene rearrangement, creating great diversity

However, T cell receptors (unlike antibodies) cannot

recognizeantigens from pathogens all by

themselves!!

T Cells Only Recognize Antigen when it is presented by another cell

Antigen presentation is done by another family of proteins called

MHC proteins

Molecular Biology of the Cell Alberts et al

Viral or bacterial proteins are digested by Cellular proteases inside the cell andpieces of them bind the MHC proteins

Molecular Biology of the Cell Alberts et al

This allows T cells to recognize HIV infected cells,for example, and even internal proteins

like reverse transcriptase can serve as antigens

Molecular Biology of the Cell Alberts et al

Here is where ourold friend CD4

comes into the picture

Let’s come back to the immune response

to HIV

People initially mount a strong immune response

However, this response ultimately fails for five reasons

However, this response ultimately fails for five reasons

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

We already discussedtwo of these

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

First, the ability to integrate into the host genome allows HIV to lurk

undetected

Second, by killing CD4+ Helper T CellsHIV ultimately disables both

antibody production andKiller T cells

What about the other three meansHIV uses for immune evasion?

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

One way HIV “hides” is by hidingits most “antigenic” regions

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

Most antibodies against the virusdo not block viral entry

Why not?

Regions of gp120 and gp41 key for viral entryare hidden until after the shape change we

discussed

Natural selection also shapesthe sequence of viral proteins

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

Remember that while reverse transcriptase is an amazing Enzyme, there was something it lacks—which was….

Remember that while reverse transcriptase is an amazing Enzyme, there was something it lacks—which was….

This has major consequences

RT makes 1 error /10,000 bp=1 error per replicated genome

And since the viral generation timeIs 2.5 days and one infected cell produces

~1010–1012 new VIRIONS each day…..

Do the numbers!

Do the numbers!

Given that billions of cells are infected per day There will be thousands of copies of

EVERY possible mutationPresent in the gene pool!!

Recombination adds to the amount of variation

Many cells are co-infected by two or more viral variants

and RT can switch between viral templates

when copying the genome

Remember these sequence based “trees” we usedto study the evolution of different HIV and SIV strains?

We can use the same approach to study the evolutionof a single virus after it infects a single person

Viral diversity in 9 AIDS patients

HIV rapidly evolves into different “strains”after the initial infection

How couldThat happen?

Can you sayNatural selection?

We start with the tremendous amountOf viral variation caused by RT errors

Now we add the selective pressureExerted by the immune response

+

In response to antibody selectionViruses with mutations

in gp120 and gp41 accumulate

T cell selection selects for changes inpeptide “epitopes” so they no longer

bind to MHC proteins

The result: despite high levels of anti-HIV antibodies

viral variants escape from the immune response

HIV also has another trick

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

Remember our discussion of Long-term non-progressors:Some are infected with a mutant HIV virus lacking the accessory gene Nef

What does Nef do?

Nef prevents infected cells from putting MHC proteinson their cell surface!

Without MHC proteins infected cells become

Invisible to T cells

HIV has even one more trick

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

Blocking Cytosine Deamination

Cytosine DeaminationCauses Mutations

Dr. Weiguo Cao website, Clemson U.

APOBEC3G is a Cytosine Deaminase Present in Resting T cells, which

HIV doesn’t infect well

APOBEC3G

APOBEC3G is incorporated into HIV virions and inhibits viral replication

by inducing hypermutation

Dr. Warner Greene's laboratory at the Gladstone Institute of Virology and Immunology

The viral accessory protein vif blocks APOBEC3G function

Vif blocks APOBEC3G incorporation into virions and

targets it for proteolytic destruction

Antigenic escape

Inaccessible epitopes

Downregulating MHC

Destruction of CD4+ T cells

Integration and latency

Blocking Cytosine Deamination

This formidable array of defense mechanismsAllows HIV to avoid being suppressed by our immune system