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

Infection

Levels

(S

ep

ara

te S

cale

s)

CD4+T-cell

HIV

CD8+T-cell

Antibodies

Years

AIDS and Death

Acute Asymptomatic

4 – 8 weeks

Flow Cytometry and Cell Sorting

Y Y

Y

YY

Y

Y

Y

Y

Y

CD45RA

CD62L

Anti-CD62L

Anti-CD45RA

Y Anti-CD8

Lymphocyte Gating

R1

Forward Scatter

Sid

e S

catt

er

R1

(How large the cell is)

(How

com

plex

the

cel

l is)

Flow CytometryGated on CD4

CD45RA

30%

5.2%

49%

CD

62L

(Brighter)(Dimmer)

T-Cell Phenotypes

Immunophenotyping

CD3+CD4+CD45+ = CD4+ T-cellCD3+CD8+CD45+ = CD8+ T-cellCD3-CD19+CD45+ = B-cells

CD3+CD4+CD28+CD45RA+ = naïve CD4+ T-cellCD3+CD4+CD28+/-CD45RA- (or RO+) = effector/memory

CD4+ T-cell

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

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

3H 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

Analysis of lymphocyte proliferation by CFSE (Carboxyfluorescein diacetate succinimidyl ester)

CFSE

Undivided1st division2nd division

Proliferative response of peripheral CD4+ T-cells to tetanus.

Media

CFSE

10 0 101 102 103 104

CD

4

100

101

102

103

104Tetanus

CFSE

100 101 102 103 104

100

101

102

103

104

4% 38%

Medium/IL-2 Tetanus/IL-2

CFSE

Healthy Control Day 7

CD

4

Proliferative response of peripheral CD4+ T-cells to tetanus.

LTNP #1 Day 7

CFSE

CD

4

Medium Tetanus

4%<1%

Proliferative response of peripheral CD4+ T-cells to p24

LTNP #1

CFSE

CD

4

Medium p24

3%<1%

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

ELISPOT Analysis For Cytokine Production

Y Y YY

Y Y YY

Y Y YY

1. Coat a well with antibodyto cytokine to be measured.

(IL-2, IL-5, IFN- etc.)

2. Add cells and antigen.Incubate overnight.

3. Cells that recognize the antigenwill become activated andsecrete cytokineswhich will be captured by the antibody.

Y

ELISPOT Analysis Cont.

Y Y YY

Y

Y Y 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 withthe enzyme. Read the plate withcomputerized ELISpot reader.

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

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

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

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 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.

51Cr Release Assay For CTL Activity

51Cr Release Assay For CTL Activity

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• Doesn’t tell you how many cells did the killing• Doesn’t identify which cells did the killing• Uses a radioisotope

ELISPOT Analysis For CTL Activity

Y Y YY

Y Y YY

Y Y YY

1. Coat a well with antibodyto IFN- or granzyme.

2. Add cells and antigen.Incubate overnight.

3. Cells that recognize the antigenwill become activated andsecrete INF- and granzymewhich will be captured by the antibody.

Y

ELISPOT Analysis For CTL Activity

Y Y YY

Y

Y Y 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 withthe enzyme. Read the plate withcomputerized ELISpot reader.

0

100

200

300

400

500

600

Env

-A

Env

-B

Env

-C

Env

-D

Env

-E

Env

-F

Env

-G

Env

-H

Env

-I

Env

-J

Env

-K

Env

-L

Env

-M

Env

-N

Gag

-A

Gag

-B

Gag

-C

Gag

-D

Gag

-E

Gag

-F

Gag

-G

Gag

-H

Nef

-A

Nef

-B

Nef

-C

Nef

-D

Pol

-A

Pol

-B

Pol

-C

Pol

-D

Pol

-E

Pol

-F

Pol

-G

Pol

-H

Pol

-I

Pol

-J

Pol

-K

Pol

-L

Pol

-M

Pol

-N

Pol

-O

Pol

-P

Rev

-A

Rev

-B

Tat

-A

Tat

-B

Vif-

A

Vif-

B

Vif-

C

Vpr

-A

Vpr

-B

Vpu

-A

Vpu

-B

Mea

n S

FC

/mill

ion

cel

ls

ELISpot Analysis of CD8+ T-cell responses to HIV During Chronic Infection

Black Bars = Freshly obtained peripheral blood CD8+ T-cellsStriped Bars = Expanded peripheral blood CD8+ T-cells

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Env Gag Nef Pol Rev Tat Vif Vpr Vpu

SF

C/M

illi

on C

D8+

/AA

Targeting of HIV proteins by CD8+ T-cells during Chronic HIV infection

Nef>Gag>Pol

Tetramer staining to quantify antigen specific CD8+ T-cells

1. Construct HLA class of the type present in the personyou are studying. Link them into tetramers foldedaround the peptide of interest.

2. Bind these tetramers to a fluorescent tag.

3. React with lymphocytes.

4. Run through a flow cytometer.

Cytotoxic T-cell

YY

MHC molecule

Antigen

FluorescentlyLabeled Strepavidin

Biotin

TCR

CD8 molecule

Tetramer Staining

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 circulatingHIV-specific CTL.

•Emergence of CTL escape mutants over time.

•Depletion of CD8+ T cells from macaques prior to infectionwith SIV, leads to higher viral loads and more profoundimmunosuppression.

•Absence of detectable HIV-specific CTL, or oligoclonal CTL responses are associated with poor clinical outcome.

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?

0

0.5

1

1.5

2

01/85 01/87 01/89 01/91 01/93 01/95

Date

% P

osi

tive

CD

8 T

cel

ls

0

0.5

1

Pro

po

rtio

n o

f S

LY

NT

VA

TL

GAG tetramer

Gamma-INF

SLYNTVATL

Donor A: CD8 response to SL9

Emergence of epitope sequence mutations

Table I. SL9-epitope and flanking amino acid sequences. Predicted amino acid sequences of the SL9-epitope and the 10 amino acids flanking each epitope are shown. Sequences were derived from plasma RNA at the indicated timepoints.

aa acid position within gag p17: 77 78 79 80 81 82 83 84 85

ID Years from

Seroconversion Proportion of clones

Variant Nomenclature S L Q T G S E E L R S L Y N T V A T L Y C V H Q R I E I K

A 0.26 9/10 SL9 A - - - - - - - - - - - - - - - - - - - - - - - - - - V - " 1/10 " A - - - E - - - - - - - - - - - - - - - - - - - - - - V - 1.52 9/10 " A - - - - - - - - - - - - - - - - - - - - - - - - - - V - " 1/10 " A - - - - - - - - - - - - - - - - - - - - - - - K - - V - 1.74 9/10 " - - - - - - - - - - - - - - - - - - - - - - - - K - - V - " 1/10 79F - - - - - - - - - - - - F - - - - - - - - - - - K - - V - 3.08 2/9 SL9 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - " 2/9 83V - - - - - - - - - - - - - - - - V - - - - - - - - - - - - " 3/9 79F - - - - - - - - - - - - F - - - - - - - - - - - - - - - - " 2/9 79F-82I - - - - - - - - - - - - F - - I - - - - - - - - - - - - - 5.51 5/11 79F-84V - - - - - - - - - K - - F - - - - V - - - - - - K - - V - " 6/11 82I-84V - - - - - - - - - K - - - - - I - V - - - - - - K - - V - 8.00 10/10 " - - - - - - - - - K - - - - - I - V - - - - - - K - - V - 8.98 10/10 " - - - - - - - - - K - - - - - I - V - - - - - - K - - V - 9.98 9/9 " - - - - - - - - - K - - - - - I - V - - - - - - K - - V - B 1.30 10/10 SL9 A - - - - - - - - - - - - - - - - - - - - - - - K - - V - 1.78 10/10 " A - - - - - - - - - - - - - - - - - - - - - - - K - - V - 3.35 9/10 " - - - - - - - - - - - - - - - - - - - - - - - - K - - - - " 1/10 " A - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3.54 12/12 84V - - - - - - - - - - - - - - - - - V - - - - - - - - - - - 4.66 9/10 79H-84V - - - - - - - - - - - - H - - - - V - - - - - - K - - V - " 1/10 82I-84V - - - - - - - - - - - - - - - I - V - - - - - - K T - V - 5.67 9/10 79F-84V - - - - - - - - - - - - F - - - - V - - - - - - K - - V - " 1/10 84V - - - - - - - - - - - - - - - - - V - - Y - - - K - - V - 6.73 6/10 " - - - - - - - - - - - - - - - - - V - - Y - - - K - - V - " 3/10 " - - - - - - - - - - - - - - - - - V - - - - - - K - - V - " 1/10 79F-84V - - - - - - - - - - - - F - - - - V - - - - - - K - - V - C 1.57 9/9 SL9 A - - - - - - - - - - - - - - - - - - - - - - - K - - V - 3.35 7/8 " - - - - - - - - - - - - - - - - - - - - - - - - K - - V - " 1/8 " - - - - - - K - - - - - - - - - - - - - - - - - K - - V - 5.77 9/9 " - - - - - - - - - - - - - - - - - - - - - - - - K - - - - 6.48 2/9 " - - - - - - - - - - - - - - - - - - - - - - - - K - - - - " 5/9 " A - - - - - - - - - - - - - - - - - - - - - - - K - - - - " 1/9 " A - - - - - - - - G - - - - - - - - - - - - - - K - - - - " 1/9 84V - - - - - - - - - - - - - - - - - V - - - - - - K - - - - 8.24 13/17 " - - - - - - - - - - - - - - - - - V - - - - - - K - - - - " 4/17 " - - - - - - - - - - - - - - - - - V - - - - - - - - - - -

Antibody Responses

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.

HIV-1 derived gp120

gp120

gp41

CD4 binding site

Gp120 is presentedas a trimer. Themonomer doesnot present theproper epitopes.

CD4 binding siteis devoid of glycosylationand relatively conservedbetween isolates but ismasked by V1V2 loopsand is in a depression.

Coreceptor bs

CD4bs

Bridging Sheet

CD4bs

V1V2 V3

V4

V5Gp41C NInner Outer

CD4bs

Bridging Sheet

Glycosylation:Silent face

Non-nuetralizingface

Trimerization

Neutralizing face

2G12

C N

Co-R bs

ANTIBODY RESPONSE TO HIV

Neutralizing antibodies are made primarily to gp120.

Group 1: Arise later in infection, recognize gp120 from a broadrange of isolates. Interfere with binding to CD4. Recognizediscontinuous 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, theseepitopes 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)

Antibody mediated inhibition of Fusion is difficult to achieve.

Changes in gp120 glycosylation allow HIV escape from Nab responses

Richman et al. PNAS 2003 vol. 100:4149

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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*

*

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*

* **

*

*

HIV and APC’s

•APC’s may exhibit altered:chemotaxis IL-1 productionantigen presentationoxidative burst responseantimycobacterial activity

•Antigen presenting cells can act asTrojan horses.

Dendritic Cells and DC-SIGN

DC-Specific, ICAM-3 Grabbing, Nonintegrin.

Interaction of DC-SIGN with ICAM-3 establishes the initialcontact of the DC with a resting T-cell.

This is important because of the low number (100-1000copies/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 theabsence of CD4.

R. Steinman Cell 2,000 100:491-494

Proposed pathways for the Transmission Of HIV-1 to T-cells.

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 enhanceHIV replication. Therefore, by responding to HIV, CD4+ T-cells can be

destroyed.

Why does the immune response fail to clear HIV?

•HIV can mutate and escape immune mediated opposition.

•Suboptimal CTL responses can be elicited.

•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?

•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?

Role of viral genes:

Tat: Extracellular Tat stimulates CD4+ and CD8+ T-cells.

Nef: Intracellular Nef appears to activate cells to promoteviral replication. Affect on cellular function?

Intracellular Nef downregulates CD4 and MHC class Imolecules. In vivo significance?

Why does the immune response fail to clear HIV?