CTL and the Controlof HIV-1 Replication

Otto Yang, M.D.

Or

Lessons learned from mixing HIV-1 and CTL

AIDS VACCINE 2007

Seattle, Washington

Evidence that CTL are protective in HIV-1 infection:

• Temporal associations with reduction in acute viremia and AIDS

• SIV-macaque model: CD8+ T cell depletion leads to sharply increased viremia, and recovery with decreased viremia

• HIV-1 sequence evolution in CTL epitopes in vivo: selective pressure (escape)

New technologies offer amazingly precise characterizations of CTL

• ELISpot: rapid, robust, validated measurements using few cells

• ICS: ability to examine phenotypes and functional markers in remarkable detail

• Such assays tell us about CTL: how many, what specificity, and what functional potentials

• How do these correlate to antiviral activity? These assays offer a detailed picture of the effector end of the CTL interaction, but what about CTL-HIV-1 interaction and the upstream events in infected cells?

Questions about the antiviral activity of HIV-1-specific CTL

• Do they kill HIV-1-infected cells?• Do they suppress HIV-1 replication (via

what mechanisms?), and what factors influence efficiency?

• Why do they fail in vivo, and how can a vaccine prevent that failure?

T Cell Receptor (On CTL)

T Cell Receptor (On CTL)

CTL recognize cells displaying foreign protein epitopes

Peptide-HLA Complex(On Infected Cell)

CTLRelease of

Cytolytic GranulesAnd

Signal for Apoptosis,Cytokines

Target Cell

HIV-1 replicates rapidly, posing a challenge to CTL

CD4+T Cell

Initial Infection

CD4+T Cell

First VirionProduced

~1 Day

Cell Death(Last Virion Produced)

~1 Day

Killing Would

Result in:Prevention of

Virion Production

Reduction ofVirion

Production

HIV-1-infected cells early in viral replication are susceptible to CTL

010203040506070

1 2 3 4Day Post-Infection

0

5

10

15

20

HIV

Tite

r (1

0^6/

ml)

Susceptibility to CTLViral Titer

O.O. Yang and R.P. Johnson et al, 1996, J Virol, 70:5799

HIV-1-specific CTL can potently inhibit viral replication

Infect CD4+T Lymphocytes

With HIV-1Culture

With CTL

CultureAlone

0.1

1

10

100

1000

10000

3 5 7 10Days

H9-B14H9-B14 + EnvCTL(B14)

Adapted from O.O. Yang and B.D. Walker et al. J. Virol 1997, 71:3120

Suppression of HIV-1 by CTLcan be irreversible

0.1

1

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10000

3 5 7 10 12 14 17 19 21 23 25Days Post Infection

Infected Cells Infected Cells + CTL

Remove CTL

Adapted from O.O. Yang and B.D. Walker et al. J. Virol 1997, 71:3120

CTL can suppress HIV-1 via cytokines and direct cytolysis

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1000000

0 4 6 8 11Day after Infection

H9H9+CTL(B14)H9-B14 + CTL(B14)H9+H9-B14+CTL(B14)

SolubleSoluble

+Lytic

Adapted from O.O. Yang and B.D. Walker et al. J. Virol 1997, 71:3120

CTL recognizing different epitopes can vary in their antiviral efficiency

-1

0

1

2

3

4

5L

og U

nits

Sup

pres

sion Control

(mismatch)RT (IV9-A2)

Gag (SL9-A2)

Nef (KL9-B60)

RT Gag NefNote that T.J. Tsomides and H.N. Eisen et al (JEM 1994, 180:1294) showed that IV9 is presented ~12/cell and SL9 is presented ~400/cell.

Unpublished data

Both Nef-mediated HLA-I downregulation and epitope kinetics

affect CTL antiviral activity

0

1

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3

4

5

Log

Uni

ts S

uppr

essi

on

Late SL9 (in Gag)/WT NefLate SL9 (in Gag)/Defective NeEarly SL9 (in Nef)/Defective N EARLY +

NO Nef effect

LATE +NO Nef effect

LATE +Nef effect

Adapted from A. Ali and O.O. Yang et al. J. Virol 2004, 78:561

WT Nef

0

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3

4

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6

7

2 4 6Day Post-Infection

The impact of Nef on CTL antiviral activity can be quantitated

Adapted from S. Adnan and O.O. Yang et al. Blood 2006, 108:3414

M20A Nef

0

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2

3

4

5

6

7

2 4 6Day Post-Infection

6.3 (no CTL)

5.5 (+CTL)

6.3 (no CTL)

0.9 (+CTL)

Nef Impact Ratio= (6.3-5.5) ÷ (6.3 -0.9) = 0.15

WT Nef

0

1

2

3

4

5

6

7

2 4 6Day Post-Infection

The impact of Nef on CTL antiviral activity can vary dramatically

Adapted from S. Adnan and O.O. Yang et al. Blood 2006, 108:3414

M20A Nef

0

1

2

3

4

5

6

7

2 4 6Day Post-Infection

6.3 (no CTL)

3.2 (+CTL)

6.3 (no CTL)

2.6 (+CTL)

Nef Impact Ratio= (6.3-3.2) ÷ (6.3 -2.6) = 0.84

Nef does not affect the antiviral activity of HLA-I C-restricted CTL

0.0

0.2

0.4

0.6

0.8

1.0

1.2N

ef Im

pact

Rat

io

B

C

A

ALL A/B ALL CAdapted from S. Adnan and O.O. Yang et al. Blood 2006, 108:3414

Other epitope factors may also affect Nef susceptibility (?)

0.0

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0.7

Nef

Impa

ct R

atio

ALLEARLY(A/B)

ALLLATE(A/B)

(Unpublished data)

0.0

0.1

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0.7

Nef

Impa

ct R

atio

B*07

B*57

A*02

ALLA*02

ALLB*07

ALLB*57

GagPolEnv

NefRev

HIV-1-specific CTL lose antiviral function with senescence

0.0

0.5

1.0

1.5

2.0

Log

Uni

ts S

uppr

essi

on

Control hTERT

Adapted from M. Dagarag and O.O. Yang et al. J Virol 2003, 77:3077

Control hTERT

Perforin

Gra

nzym

e B

From O.O. Yang and C. Uittenbogaart et al. Virol 2004, 332:16

Summary of factors determining CTL antiviral efficiency

• Epitope levels (adequacy for recognition)• Epitope kinetics (timing of recognition in

relation to viral production)• Impact of Nef-mediated HLA-I

downregulation• Effector status of CTL• What is the relative importance of these

factors???

0 10 20 30 40 50 60 70 80 90

RPAEPVPLQL

-S--------

--T-------

--E-------

---Q------

--------H-

-------F--

--V-------

--E----F--

-------FH-

--Q-----L-

Epi

tope

Seq

uenc

e

CTL killing of peptide-loaded versus HIV-infected cell

Peptide 10µg/mlInfected C

An example that antiviral assays can give different results than standard CTL assays

(Implications for escape and cross-clade studies?)

0 10 20 30 40 50 60 70 80 90

RPAEPVPLQL

-S--------

--T-------

--E-------

---Q------

--------H-

-------F--

--V-------

--E----F--

-------FH-

--Q-----L-

Epi

tope

Seq

uenc

e

CTL killing of peptide-loaded versus HIV-infected cell

Peptide 10µg/mlInfected C

Clade A1 consensusClade A2 and F2 consensus

Clade F1 consensus

MS Bennett and OO Yang, Submitted

CTL detection does not predict virus suppression

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3 4 5 6 7Days

Control +CTL

RPAEPVPLQL(Consensus B/C/G)

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3 4 5 6 7Days

Control +CTL

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3 4 5 6 7Days

Control +CTL

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3

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5

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3 4 5 6 7Days

Control +CTL

-S--------(Consensus A1)

--T-------(Consensus A2/F2)

--E-------(Consensus F1)

MS Bennett and OO Yang, Submitted

Implications for analysis of current vaccine trials?

• ELISpot is the major validated surrogate marker for CTL immunogenicity of vaccines

• Most likely, ELISpot responses are necessary but not sufficient to reflect the antiviral activity of CTL (recognition of infected cells)

• If current vaccine trials fail to demonstrate clinical benefit despite “immunogenicity,” it may be premature to discount a CTL-based vaccine approach based on lack of protection in vaccine “responders”

A more important question about HIV-1-specific CTL:

Why do they fail in vivo?

Multiple mechanisms have been observed and proposed to explain failure in vivo

• Abnormal differentiation and activation

• Exhaustion/senescence• Hypofunctional profile• Lack of CD4 help• Epitope escape mutation

Can a single process explain these diverse events (Ockham’s razor)???

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1000000

0 5 10

Day

HIV (SLYNAVATL) + CTL

Single amino acid changes in epitopes can allow HIV-1 to completely avoid CTL

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0 5 10

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HIV (SLYNTVATL) + CTL

HIV-1Infection

Immunodominance of Poorly Conserved

Epitopes

A hypothesis….Early Escape andPoor Containment

Early Massive Irreversible CD4+

T Cell Loss

Original Antigenic

Sin

Decreasing CTL Quantity and Quality

Eventual Complete Failure

Abnormal CTL Activation and Differentiation

Ongoing Escape

Ongoing HIV-1 Replication

HIV-1Infection

Immunodominance of Poorly Conserved

Epitopes

A hypothesis….

Early Reduction of HIV-1

Preserved CD4+ T

Helper Cells

Effective CTL

Containment of HIV-1

Targeting of Conserved Epitopes

Vaccine Priming

What strategies can prevent escape?

All randomly generated epitope mutants

Viable mutants

Unrecognizable mutantsEscape

Mutants

A mutation must be both non-recognized and viable to allow escape

All randomly generated epitope mutants

Viable mutants

Unrecognizable mutants

NoEscapeMutants

Bypassing immunodominance to focus CTL on very constrained epitopes?

All randomly generated epitope mutants

Viable mutants

EscapeMutants

Viable mutants

Unrecognizable mutants

Focus immunity on constrained sequences?

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

From entropy calculations generously provided by B Korber

AcknowledgmentsS AdnanA AliA BalamuruganMS BennettMJ LewisHL NgPT Nguyen Sarkis

C BranderA Trocha

BD Jamieson

RP JohnsonSA KalamsBD Walker

D Wick

Bette KorberNIH: AI043203 and AI051970