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

2. Your body has evolved complex mechanisms of recognizing “non-self” and fighting against it

3. The Immune System is the Third Line of Defense Against Infection

4. Antibodies are Produced by B Lymphocytes (B cells to their friends)

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

6. Overview of Human Immunity: Adaptive and Innate

7. Lymphocyte Origins
16-22

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

9. Some definitions are in order
Antigen
A substance produced by a pathogen
(e.g., protein, complex sugar)
capable of producing an immune response

10. Some definitions are in order
Antibodies
Protein molecules (immunoglobulins)
produced by B lymphocytes
to help eliminate an antigen

11. Molecular Biology of the Cell Alberts et al
B cells
Make
Antibodies
In response to
antigens
Molecular Biology of the Cell Alberts et al

12. 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

13. Antibodies can also direct phagocytosis of pathogens
Molecular Biology of the Cell Alberts et al

14. Cytotoxic (Killer) T Cells Recognize, Attack and Kill Virus-Infected Cells
CELLS alive!

15. Let’s focus first on antibodies
Molecular Biology of the Cell Alberts et al

16. Antibodies are proteins that have evolved
to recognize molecules from pathogens
Molecular Biology of the Cell Alberts et al

17. These molecules from pathogens are called Antigens

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

19. 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

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

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

22. This interaction is VERY specific
Antigen residues
at the interface
= epitope
Epitopes are
typically ~5
residues long

23. This interaction is VERY specific
hemagglutinin
antibody
Space-filling mode
Grey now = mainchain
of hemagglutinin
Epitopes reside in
turns and loops

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

25. 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

26. 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

27. 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

28. requires rearranging the DNA
Molecular Biology of the Cell Alberts et al

29. requires rearranging the DNA
Molecular Biology of the Cell Alberts et al

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

31. 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.

32. 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.

33. 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

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

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

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

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

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

39. However, T cell receptors (unlike antibodies) cannot recognize antigens from pathogens all by themselves!!

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

41. Antigen presentation is done by another family of proteins called MHC proteins

42. 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

43. 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

44. 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

45. Let’s come back to the immune response to HIV

46. People initially mount a strong immune response

47. However, this response ultimately fails for five reasons

48. However, this response ultimately fails for five reasons
Integration and latency
Destruction of CD4+ T cells
Inaccessible epitopes
Antigenic escape
Downregulating MHC

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

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

51. Second, by killing CD4+ Helper T Cells HIV ultimately disables both antibody production and Killer T cells

52. 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

53. 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

54. 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

55. Why not?

56. 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.

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

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

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

60. 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…..

61. Do the numbers!

62. 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!!

63. 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

64. Remember these sequence based “trees” we used
to study the evolution of different HIV and SIV strains?

65. We can use the same approach to study the evolution
of a single virus after it infects a single person

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

67. How could
That happen?

68. Can you say
Natural selection?

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

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

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

72. 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

73. The result: despite high levels of anti-HIV antibodies viral variants escape from the immune response

74. HIV also has another trick
Integration and latency
Destruction of CD4+ T cells
Inaccessible epitopes
Antigenic escape
Downregulating MHC

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

76. What does Nef do?

77. Nef prevents infected cells
from putting MHC proteins
on their cell surface!

78. Without MHC proteins
infected cells become
Invisible to T cells

79. HIV has even one more trick
Integration and latency
Destruction of CD4+ T cells
Inaccessible epitopes
Antigenic escape
Downregulating MHC
Blocking Cytosine Deamination

80. Cytosine Deamination Causes Mutations
Dr. Weiguo Cao website, Clemson U.

81. APOBEC3G is a Cytosine Deaminase Present in Resting T cells, which HIV doesn’t infect well

82. 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

83. The viral accessory protein vif blocks APOBEC3G function
Vif blocks APOBEC3G incorporation into virions and targets it for proteolytic destruction

84. 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