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

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 Molecular Biology of the Cell Alberts et al

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 Molecular Biology of the Cell Alberts et al

These molecules from pathogens are called Antigens

The variable and constant regions of antibodies are related = Ig domains Molecular Biology of the Cell Alberts et al

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

Bound to the “antigen” = influenza hemagglutinin Here is the antibody Bound to the “antigen” = influenza hemagglutinin Hemagglutinin Human antibody

The antibody recognizes the antigen by a lock-and-key fit Rotate ~90 Add all atoms

This interaction is VERY specific 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

You can generate antibodies against HIV like you do against other viruses

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

just to make antibodies. 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

just to make antibodies. What’s the solution? 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

Molecular Biology of the Cell Alberts et al The result: an antibody light chain Molecular Biology of the Cell Alberts et al

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 This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.   K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both.

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 Isn't that amazing! This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.   K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both.

Other mechanisms further increase antibody diversity This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.   K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both. 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

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

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 creating great diversity by gene rearrangement, creating great diversity Molecular Biology of the Cell Alberts et al

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

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

Molecular Biology of the Cell Alberts et al Here is where our old friend CD4 comes into the picture Molecular Biology of the Cell Alberts et al

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

We already discussed two of these Integration and latency Destruction of CD4+ T cells Inaccessible epitopes Antigenic escape Downregulating MHC

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

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

Most antibodies against the virus do not block viral entry HIV-infected patients and monkeys experimentally infected with SIV typically generate high levels of circulating antibodies that recognize both linear and conformational epitopes in gp120 and gp41. These antibodies generally react well with envelope monomers by ELISA and with gp120 and gp41 proteins in western blots. However, they react poorly, if at all, with the fully assembled complex on virions and the surface of infected cells HIV-infected patients and monkeys experimentally infected with SIV typically generate high levels of circulating antibodies that recognize both linear and conformational epitopes in gp120 and gp41. These antibodies generally react well with envelope monomers by ELISA and with gp120 and gp41 proteins in western blots. However, they react poorly, if at all, with the fully assembled complex on virions and the surface of infected cells

Why not?

Regions of gp120 and gp41 key for viral entry are hidden until after the shape change we discussed acutely infectious viral agents are quite uniformly neutralization-sensitive. This is a direct reflection of the differences in propagation strategies. Acutely infectious agents work around the immune response by being highly contagious, relying on high levels of replication and transmission before the host mounts an immune attack. The replication strategy of HIV and SIV, involving a prolonged period of continuous replication in each infected individual, requires an envelope complex structure that sacrifices some efficiency in inherent infectivity in order to achieve a level of neutralization resistance not seen with the acute viruses.

Natural selection also shapes the sequence of viral proteins Integration and latency Destruction of CD4+ T cells Inaccessible epitopes Antigenic escape Downregulating MHC

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 ~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 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

HIV rapidly evolves into different “strains” after the initial infection Viral diversity in 9 AIDS patients

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 + 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 extraordinarily high ratio of nonsynonomous to synonomous substitutions within the variable regions of the envelope protein (54). Specifically, more than 95% of all the nucleotide changes in the gp120 envelope protein resulted in an amino acid change (in the absence of selection, random substitutions are expected to lead to nonsynonomous changes only about 70% of the time 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 Integration and latency Destruction of CD4+ T cells Inaccessible epitopes Antigenic escape Downregulating MHC

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

This formidable array of defense mechanisms Allows HIV to avoid being suppressed by our immune system Integration and latency Destruction of CD4+ T cells Inaccessible epitopes Antigenic escape Downregulating MHC Blocking Cytosine Deamination