The branch that breaks Is called rotten, but Wasn’t there snow on it? Bartolt Brecht Haiti after a hurricane

Your body has evolved complex mechanisms 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 to their 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 Antibodies In response to antigens

Molecular Biology of the Cell Alberts et al These antibodies can bind to and “neutralize” Viruses or can direct immune attack of virus-infected cells

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

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 of antibodies are related = Ig domains

Constant Region Hypervariable Region Light Chain Heavy Chain Antigen Binding Region Let’s use as an example an antibody that recognizes a protein on the surface of flu (influenza) virus courses.washington.edu/medch401/pdf_text/401_07_lect2.ppt

Hemagglutinin Here is the antibody Bound 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 = mainchain of 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

Other mechanisms further increase antibody diversity Molecular Biology of the Cell Alberts et al

When a pathogen enters the body it stimulates proliferation of the specific B Cells that recognize its Antigens

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

CELLS alive! OK, that explains antibodies and B cells but what about us?

Molecular Biology of the Cell Alberts et al T cells carry antibody-related proteins on their plasma membranes called T cell receptors

Molecular Biology of the Cell Alberts et al T cell receptors are also assembled by gene rearrangement, creating great diversity

However, T cell receptors (unlike antibodies) cannot recognize antigens 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 and pieces 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 our old 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

Antigenic escape Inaccessible epitopes Downregulating MHC Destruction of CD4+ T cells Integration and latency

We already discussed two 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 Cells HIV ultimately disables both antibody production and Killer T cells

What about the other three means HIV uses for immune evasion? Antigenic escape Inaccessible epitopes Downregulating MHC Destruction of CD4+ T cells Integration and latency

One way HIV “hides” is by hiding its most “antigenic” regions Antigenic escape Inaccessible epitopes Downregulating MHC Destruction of CD4+ T cells Integration and latency

Most antibodies against the virus do not block viral entry

Why not?

Regions of gp120 and gp41 key for viral entry are hidden until after the shape change we discussed

Natural selection also shapes the 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 time Is 2.5 days and one infected cell produces ~10 10 –10 12 new VIRIONS each day…..

Do the numbers!

Given that billions of cells are infected per day There will be thousands of copies of EVERY possible mutation Present 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 used to study the evolution of different HIV and SIV strains?

We can use the same approach to study the evolution of 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 could That happen?

Can you say Natural selection?

We start with the tremendous amount Of viral variation caused by RT errors

Now we add the selective pressure Exerted by the immune response +

In response to antibody selection Viruses with mutations in gp120 and gp41 accumulate

T cell selection selects for changes in peptide “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 proteins on 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 Deamination Causes 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 mechanisms Allows HIV to avoid being suppressed by our immune system