Immune Response to HIV

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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

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AIDS and Death
Acute
Asymptomatic
Infection
CD8 T-cell
HIV
Levels (Separate Scales)
Antibodies
CD4 T-cell
Years
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Flow Cytometry and Cell Sorting
CD45RA
CD62L
Anti-CD62L
Y
Anti-CD45RA
Y
Anti-CD8
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Lymphocyte Gating
Side Scatter
(How complex the cell is)
R1
Forward Scatter
(How large the cell is)
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Flow Cytometry
Gated on CD4
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CD62L
5.2
(Brighter)
(Dimmer)
CD45RA
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T-Cell Phenotypes
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Immunophenotyping
CD3CD4CD45 CD4 T-cell CD3CD8CD45 CD8
T-cell CD3-CD19CD45 B-cells CD3CD4CD28CD45
RA naïve CD4 T-cell CD3CD4CD28/-CD45RA-
(or RO) effector/memory CD4 T-cell
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CD4 T-cell Responses To HIV

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

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

• Pros
• Relatively easy
• Well accepted method
• Cons
• Measures relative amounts of proliferation
• Doesnt tell you how many cells
• Doesnt identify which cells

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Analysis of lymphocyte proliferation by CFSE
(Carboxyfluorescein diacetate succinimidyl ester)
Undivided
1st division
2nd division
CFSE
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Proliferative response of peripheral CD4
T-cells to tetanus.
Healthy Control Day 7
Medium/IL-2
Tetanus/IL-2
CD4
CFSE
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Proliferative response of peripheral CD4
T-cells to tetanus.
LTNP 1 Day 7
Tetanus
Medium
4
lt1
CFSE
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Proliferative response of peripheral CD4
T-cells to p24
LTNP 1
Medium
p24
3
lt1
CFSE
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CFSE ASSAY

• Pros
• Gives percentage of cells that proliferated
• Allows identification of cells that proliferated
• Detects the number of times cells proliferated
• Doesnt use radioactive isotopes
• Cons
• A little more complex to perform
• CFSE can have some low level toxicity to cells
• Requires more skill to interpret

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ELISPOT Analysis For Cytokine Production
Y
Y
Y
Y
Y

• Coat a well with antibody
• to cytokine to be measured.
• (IL-2, IL-5, IFN-g 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.
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ELISPOT Analysis Cont.
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.
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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 doesnt allow phenotyping of secreting
  cells

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CD4 T-cell Response To HIV cont.
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
Mandell Mildvan I AIDS
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CD8 T-cells Two potential mechanisms for viral
control.
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CTL Responses To HIV

• CTL responses are measured by
• 51Cr release assay The gold standard for
  killing. While it gives relative levels of CTL
  activity, it doesnt 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 persons HLA type.

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51Cr Release Assay For CTL Activity
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51Cr Release Assay For CTL Activity
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51Cr 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
• Doesnt tell you how many cells did the killing
• Doesnt identify which cells did the killing
• Uses a radioisotope

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ELISPOT Analysis For CTL Activity
Y
Y
Y
Y
Y

• Coat a well with antibody
• to IFN-g or granzyme.

2. Add cells and antigen. Incubate overnight.
3. Cells that recognize the antigen will become
activated and secrete INF-g and granzyme which
will be captured by the antibody.
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ELISPOT Analysis For CTL Activity
4. Wash away cells.
5. Add a second antibody to IFN-g 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.
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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
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Targeting of HIV proteins by CD8 T-cells during
Chronic HIV infection
3.5
3.0
2.5
2.0
SFC/Million CD8/AA
1.5
1.0
0.5
0.0
Env
Gag
Nef
Pol
Rev
Tat
Vif
Vpr
Vpu
NefgtGaggtPol
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Tetramer staining to quantify antigen specific
CD8 T-cells

• 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.
• React with lymphocytes.
• Run through a flow cytometer.

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

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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?

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Donor A CD8 response to SL9
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Emergence of epitope sequence mutations
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Antibody Responses
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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.

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HIV-1 derived gp120
Gp120 is presented as a trimer. The monomer
does not present the proper epitopes.
gp120
CD4 binding site
CD4 binding site is devoid of glycosylation and
relatively conserved between isolates but
is masked by V1V2 loops and is in a depression.
gp41
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Gp41
V5
C
N
C N
Inner
Outer
V4
V1V2
V3
Co-R bs
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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 carbohydr
ate in the outer domain. Rare but broadly
neutralizing. (2G12)
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Antibody mediated inhibition of Fusion is
difficult to achieve.
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Changes in gp120 glycosylation allow HIV escape
from Nab responses
Richman et al. PNAS 2003 vol. 1004149
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Changes in the ability of HIV infected
individuals to neutralize HIV over time.
Richman et al. PNAS 2003 vol. 1004149
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HIV and APCs

• APCs may exhibit altered
• chemotaxis
• IL-1 production
• antigen presentation
• oxidative burst response
• antimycobacterial activity
• Antigen presenting cells can act as
• Trojan horses.

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Dendritic Cells and DC-SIGN
DC-Specific, ICAM-3 Grabbing, Nonintegrin. Intera
ction 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.
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Proposed pathways for the Transmission Of HIV-1
to T-cells.
R. Steinman Cell 2,000 100491-494
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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.

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Why does the immune response fail to clear HIV?

• HIV can mutate and escape immune mediated
  opposition.
• Suboptimal CTL responses can be elicited.

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Why does the immune response fail to clear HIV?

• 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.

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Why does the immune response fail to clear HIV?

• APCs 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.

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Why does the immune response fail to clear HIV?
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?