1. Immune Response to HIV Assays to measure immune responses CD4: Helper T-cell responses CTL: Cytotoxic T-cell responses B-cell: Antibody responses APC: Antigen Presenting Cells

2. Infection Levels (Separate Scales) CD4+ T-cell HIV CD8+ T-cell Antibodies Years AIDS and Death AcuteAsymptomatic 4 – 8 weeks

3. Flow Cytometry and Cell Sorting Y Y Y Y Y Y Y Y Y Y CD45RA CD62L Anti-CD62L Anti-CD45RA Y Anti-CD8

5. Lymphocyte Gating Forward Scatter Side Scatter R1 (How large the cell is) (How complex the cell is)

6. Flow Cytometry Gated on CD4 CD45RA 30% 5.2% 49% CD62L (Brighter) (Dimmer)

7. T-Cell Phenotypes

8. Immunophenotyping CD3+CD4+CD45+ = CD4+ T-cell CD3+CD8+CD45+ = CD8+ T-cell CD3-CD19+CD45+ = B-cells CD3+CD4+CD28+CD45RA+ = naïve CD4+ T-cell CD3+CD4+CD28+/-CD45RA- (or RO+) = effector/memory CD4+ T-cell

9. CD4+ T-cell Responses To HIV CD4+ T-cell responses to antigens are usually indirectly measured by proliferation (cell division). 3 H-Thy uptake CFSE Cytokine production is another measure of activation Eliza ELISpot Flow cytometry: Polychromatic flow

10. 3 H-thymidine uptake for measuring CD4+ T-cell proliferation.

11. 3 H Thymidine Pros: Relatively easy Well accepted method Cons: Measures relative amounts of proliferation Doesn’t tell you how many cells Doesn’t identify which cells

12. Analysis of lymphocyte proliferation by CFSE ( Carboxyfluorescein diacetate succinimidyl ester) CFSE Undivided1 st division2 nd division

13. Proliferative response of peripheral CD4 + T-cells to tetanus. Medium/IL-2Tetanus/IL-2 CFSE Healthy Control Day 7 CD4

14. Proliferative response of peripheral CD4 + T-cells to tetanus. LTNP #1 Day 7 CFSE CD4 Medium Tetanus 4% <1%

15. Proliferative response of peripheral CD4 + T-cells to p24 LTNP #1 CFSE CD4 Medium p24 3%<1%

16. CFSE ASSAY Pros: Gives percentage of cells that proliferated Allows identification of cells that proliferated Detects the number of times cells proliferated Doesn’t use radioactive isotopes Cons: A little more complex to perform CFSE can have some low level toxicity to cells Requires more skill to interpret

17. ELISPOT Analysis For Cytokine Production Y Y YY Y Y YY Y Y YY 1.Coat a well with antibody to cytokine to be measured. (IL-2, IL-5, IFN-  etc.) 2. Add cells and antigen. Incubate overnight. 3. Cells that recognize the antigen will become activated and secrete cytokines which will be captured by the antibody. Y

18. ELISPOT Analysis Cont. Y Y YY Y YY Y Y Y YY 4. Wash away cells. 5. Add a second antibody to the cytokine, linked to an enzyme. 6. Develop the plate by adding a colored substrate to react with the enzyme. Read the plate with computerized ELISpot reader.

19. ELISpot ASSAY Pros: Relatively easy Highly quantitative. Allows determination of the number of responding cells. Dot size can indirectly allow relative assessment of cytokine amounts (-, +, ++, etc.) Cons: Currently doesn’t allow phenotyping of secreting cells

20. CD4+ T-cell responses are predictive of disease progression. In most individuals, the following pattern is observed: CD4+ T-cell responses decline at various stages: response to HIV and recall antigens (early) response to alloantigens(mid) response to mitogens(late) expression of IL-2 receptor (CD25) In addition, there is aberrant cytokine production production of IFN-g, IL-2 production of IL-4, IL-10 CD4+ T-cell Response To HIV cont. Mandell & Mildvan I AIDS

22. CD8+ T-cells: Two potential mechanisms for viral control.

23. CTL Responses To HIV CTL responses are measured by 51 Cr release assay: The “gold standard” for killing. While it gives relative levels of CTL activity, it doesn’t quantify the number of epitope specific cells. ELISpot: Does not directly demonstrate killing, but does allow quantification of epitope specific cells. Tetramer staining: Quantifies epitope specific CD8+ T-cells and allows for phenotypic analysis. Restricted to one epitope and requires knowledge of person’s HLA type.

24. 51 Cr Release Assay For CTL Activity

26. 51 Cr Release Assay Pros: The only assay that measures killing with the caveat that flow-based assays are being developed Cons: Measures relative amounts of killing Doesn’t tell you how many cells did the killing Doesn’t identify which cells did the killing Uses a radioisotope

27. ELISPOT Analysis For CTL Activity Y Y YY Y Y YY Y Y YY 1.Coat a well with antibody to IFN-  or granzyme. 2. Add cells and antigen. Incubate overnight. 3. Cells that recognize the antigen will become activated and secrete INF-  and granzyme which will be captured by the antibody. Y

28. ELISPOT Analysis For CTL Activity Y Y YY Y YY Y Y Y YY 4. Wash away cells. 5. Add a second antibody to IFN-  or granzyme, linked to an enzyme. 6. Develop the plate by adding a colored substrate to react with the enzyme. Read the plate with computerized ELISpot reader.

29. ELISpot Analysis of CD8+ T-cell responses to HIV During Chronic Infection Black Bars = Freshly obtained peripheral blood CD8+ T-cells Striped Bars = Expanded peripheral blood CD8+ T-cells

30. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 EnvGagNefPolRevTatVifVprVpu SFC/Million CD8+/AA Targeting of HIV proteins by CD8+ T-cells during Chronic HIV infection Nef>Gag>Pol

31. Tetramer staining to quantify antigen specific CD8+ T-cells 1.Construct HLA class of the type present in the person you are studying. Link them into tetramers folded around the peptide of interest. 2. Bind these tetramers to a fluorescent tag. 3.React with lymphocytes. 4.Run through a flow cytometer.

32. Cytotoxic T-cell Y Y MHC molecule Antigen Fluorescently Labeled Strepavidin Biotin TCR CD8 molecule Tetramer Staining

33. HIV Specific CTL are critical for control of HIV Replication CTL responses are made to various epitopes on: Gag, RT, Env, Pol, Nef, Vif, Vpr Inverse correlation between viral load and levels of circulating HIV-specific CTL. Emergence of CTL escape mutants over time. Depletion of CD8+ T cells from macaques prior to infection with SIV, leads to higher viral loads and more profound immunosuppression. Absence of detectable HIV-specific CTL, or oligoclonal CTL responses are associated with poor clinical outcome.

34. CTL fail to eliminate HIV-1 Many chronically infected individuals have vigorous HIV-1-specific CTL responses yet they almost always fail to adequately suppress the virus. Why?  Epitope escape?  CTL Exhaustion?  Suboptimal CTL?

35. Donor A: CD8 response to SL9

36. Emergence of epitope sequence mutations

38. Antibody Responses

39. General Properties of Anti-viral Antibodies Can be generated to any accessible portion of the virus. Effective in blocking entry (neutralizing) if directed to viral receptors such as gp120 of HIV. Can block fusion (neutralizing) if antibody (Ab) binds to fusion protein such as gp41 of HIV. Can effect clearance of virus if it binds the virus and then binds Fc receptors on monocytes and macrophages. Can also bind complement and kill enveloped viruses. Most effective if they are present at the site of viral entry.

40. HIV-1 derived gp120 gp120 gp41 CD4 binding site Gp120 is presented as a trimer. The monomer does not present the proper epitopes. CD4 binding site is devoid of glycosylation and relatively conserved between isolates but is masked by V1V2 loops and is in a depression.

42. Coreceptor bs CD4bs Bridging Sheet CD4bs V1V2 V3 V4 V5 Gp41 CN Inner Outer CD4bs Bridging Sheet Glycosylation: Silent face Non-nuetralizing face Trimerization Neutralizing face 2G12 C N Co-R bs

43. ANTIBODY RESPONSE TO HIV Neutralizing antibodies are made primarily to gp120. Group 1: Arise later in infection, recognize gp120 from a broad range of isolates. Interfere with binding to CD4. Recognize discontinuous epitopes known as the CD4bs epitopes. Group 2: Are directed to epitopes induced by gp120 binding to CD4. These are located near the conserved gp41 structures import- ant for chemokine receptor interactions. However, these epitopes are poorly exposed prior to CD4 binding. (17b) (2F5 is the only confirmed antibody to bind gp41) Group 3: Recognizes a conserved epitope most likely conserved carbohydrate in the outer domain. Rare but broadly neutralizing. (2G12)

44. Antibody mediated inhibition of Fusion is difficult to achieve.

45. Changes in gp120 glycosylation allow HIV escape from Nab responses Richman et al. PNAS 2003 vol. 100:4149

46. Changes in the ability of HIV infected individuals to neutralize HIV over time. Richman et al. PNAS 2003 vol. 100:4149

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50. HIV and APC’s APC’s may exhibit altered: chemotaxis IL-1 production antigen presentation oxidative burst response antimycobacterial activity Antigen presenting cells can act as Trojan horses.

51. Dendritic Cells and DC-SIGN DC-Specific, ICAM-3 Grabbing, Nonintegrin. Interaction of DC-SIGN with ICAM-3 establishes the initial contact of the DC with a resting T-cell. This is important because of the low number (100-1000 copies/cell) of MHC-peptide ligands on the DC. This enhanced binding allows the T-cell to scan the surface of the DC. DC-SIGN also binds the glycan-rich HIV-1 envelope in the absence of CD4.

52. R. Steinman Cell 2,000 100:491-494 Proposed pathways for the Transmission Of HIV-1 to T-cells.

53. Why does the immune response fail to clear HIV? HIV integrates into the host genome. Therefore, to eliminate HIV, infected cells must be killed. Host factors can paradoxically enhance HIV replication. Therefore, by responding to HIV, CD4+ T-cells can be destroyed.

54. Why does the immune response fail to clear HIV? HIV can mutate and escape immune mediated opposition. Suboptimal CTL responses can be elicited.

55. Sugar coating (glycosylation) and folding of gp120 protects against Ab recognition. Critical binding sites on gp41 are revealed for only a short period of time. Why does the immune response fail to clear HIV?

56. APC’s may exhibit altered functions diminishing their ability to elicit immune responses. Antigen presenting cells can act as Trojan horses, spreading HIV to CD4+ T-cells as they begin to respond to antigen. Why does the immune response fail to clear HIV?

57. Role of viral genes: Tat: Extracellular Tat stimulates CD4+ and CD8+ T-cells. Nef: Intracellular Nef appears to activate cells to promote viral replication. Affect on cellular function? Intracellular Nef downregulates CD4 and MHC class I molecules. In vivo significance? Why does the immune response fail to clear HIV?