2014 “Towards an HIV Cure” symposiumMelbourne

Induction and Clearance of Latent HIV Infection:

an Ex-Vivo Assessment of Immune Effectors using Cells from ART-treated

Patients

DM Margolis1, C Garrido1, JM Sung1, S Lam2, R Bateson1, B Allard1, N Dahl1, CR Cruz2, P Castillo-Caro3, MT Ngo3, J Kuruc1,

A Crooks1, CM Rooney3, CM Bollard2, and NM Archin1

1University of North Carolina Chapel Hill, NC; 2Children’s National Medical Center, Washington, DC; 3Baylor College of Medicine,

Houston, Texas

Model systems to assess clearance

Primary cell systems Cytotoxic T cells NK cellso ADCC antibodieso Immunotoxinso Selective apoptosis inducers

Why Ex-vivo Expanded CTLs

Bypasses impaired immune response Precisely controlled quantity & timing of

administration CTLs can persist Safe and effective for treatment of viral

infections in oncology patientsAM Leen, et al. Nat Med. 2006

Bollard, et al. Blood. 2007

Mature DC + gag/ pol/ nef

++HIV-CTLsT cell

Immature DC

PBMC

freeze

PHAblast

IL-7 IL-15 IL-2IL-12IL-15

CD80/86

4-1BBL

CD32

+ K562

ART to prevent In vitro spread

Cath Bollard, DC Children’sClio Rooney, Baylor and PACT

Ex vivo Expansion of HIV-specific T cells

HXTCs

HXTCs are a Mixture of Phenotypes

0 1 2 3 4 5 6 70

10

20

30

40

50

60

70

80

90

100

%L

ymp

ho

cyte

s

82% CD8+ T cells19% CD4+ T cells80% EM T cells13% CM T cells

IFNγ Releaseto Cognate Peptides

HXTCs Inhibit Productively Infected Cells

CD8 deplete patient PBMCsActivate 2-3 days

Superinfect w/ virus via spinoculation

+ autologous unexpanded CD8 or expanded HIV-CTLs (or no effector control)

Co-culture x 7 daysMedia change q3-4 daysSupernatant harvested for p24 ELISA Yang, et al. JID 2012

Freel, et al. J Virol. 2012

wash

Plate in triplicate

HIV-CTLs

HXTCs Inhibit Autologous Reservoir Virus

E:TE:T

E:T

E:T

Patient 492

No effectors 1:100

20

40

60

80

100

120

140

Patient 532

No effectors 1:10 1:10

20

40

60

80

100

120

Patient 250

No effectors 1:10 1:10

20

40

60

80

100

120 No EffectorsCD8HXTC

Patient 425

No effectors 1:100

20

40

60

80

100

120

HIV

p24

(%

of

no e

ffect

or a

t da

y 7)

Ex-Vivo Latency Clearance Assay:A modified quantitative viral outgrowth Assay

Resting CD4 Negative selection CD8, CD14, CD16, CD19, CD56, glycophorin A

CD8 HXTCs

InductionPHA/IL2

or VOR

Plate: 0.5-1x106/well, 12 wells group

PBMCsNo

Effectorsor or

Add CTLsCo Cx

24H

Add AlloFeeders

CoCxx2

Measure p24at

Day 15

washwash

Media changes q3-4 days

Cx with ARVs24H

CTL Expansion CD8 negative selection

HXTCs Clear Infected Cells Emerging from Latency

PHA Stimulation VOR Induction

No Effectors CD8 HXTC0

1

2

3

4

5

6

7

8

#w

ell

s p

os

itiv

e

(ou

t o

f 1

2 t

ota

l)

425 532 2500

20

40

60

80

100

120

% v

ira

l re

co

ve

ry

Et 1:10 Et 1:10

p <0.03 Wilcoxon signed rank p <0.02 t test

VOR does not Impair CD8 Antiviral Activity at Physiogically Relevant Exposures

HIV

p2

4 (

ng

/ml)

Viral inhibitionassay at E:T

ratio 1:1

hours

B.

Control 24 48 720

20

40

60

80

100No CD8 Control

335nM500nM1000nM

No VOR

Patient 532

Why NKs to target residual HIV

Crucial innate immune effectors against viral infections Do not recognize specific antigens nor require prior antigen sensitization Function is balanced by inhibitory and activating receptors

Kill cells by release of granzymes and perforins, which causes apoptosis NK cells may help control HIV-1 infection Memory NK cells may also provide a more effective antiviral

response

NK function may be augmented by clinically applicable cytokines

VIRAL INHIBITION ASSAY

PBMCsNegativ

e isolation

NK cells

CD4+T cells

PHA 24h

Super-infection

(autologous reservoir

virus)

HIV-CD4+ cells

+/- effectors

Unstimulated NKs

IL-2 /IL-15stimulated NKs

HIV-p24

7 days

Ne

ga

tive

is

ola

tion

No effectors

≠ E:T ratios

VIRAL INHIBITION ASSAY

1:1

1:1

0

1:1

00

1:1

1:1

0

1:1

00

1:1

1:1

0

1:1

00

resting 100 U/mL 25 ng/mLTAR-GETS

ALONE

NK NK + IL2 NK + IL-15

0

20

40

60

80

100

120 % HIV replication Day 7 autologous virus

1:1 1:10 1:100

1:1 1:10 1:100

1:1 1:10 1:100

NK cells

CD4+Targets

only IL2-stimulated NK cells IL15-stimulated NK cells

†

†

†

†

†

†

†: p <0.01

LATENCY CLEARANCE ASSAY

PBMCsNegativ

e isolation

NK cells

Resting CD4+T

cells

ARV 24hStimulation

CD4+T cells

+/- effectors

Add feeders

Unstimulated NKs

IL-2 stimulated NKs

Measure number of positive wells for

p24 at day 15

24h

24h

Ne

ga

tive

is

ola

tion

No effectors

Compare number of positive wells

with/without effectors

LATENCY CLEARANCE ASSAY

PHA reactivation VOR reactivation

VOR

Positive trends but more assays needed

IMPACT OF VOR ON NK FUNCTION

- NKs were treated with or without 335 nM VOR overnight - For degranulation cells 4 hours in the presence of K562 cell targets

VOR did not impair NK cytotoxicity or antiviral activity

Targets alone

1:1 1:10 1:1000

20

40

60

80

100

120

140

100

38.76

87.1181.20

100

38.00

79.04

102.81

NK resting NK + SAHA (335 nM)

NK resting NK+SAHA05

101520253035404550

20.34 22.64

%CD56+CD107a+

Vir

al i

nh

bit

ion

ass

ays

Deg

ranu

lation

VOR

Summary

Polyclonal HXTCs with activity against multiple peptides can be generated in clinically relevant numbers

HXTCs and cytokine-treated NK cells show enhanced antiviral activity Using both lab strain JR-CSF virus and autologous reservoir virus

NK cells and HXTCs reduce viral recovery from resting CD4 cells reactivated with PHA/IL-2, demonstrating a potential to clear latent HIV infection ex-vivo Preliminary results also show reduction in viral recovery

from resting CD4 cells reactivated with VOR, and no adverse effect on function at relevant VOR exposures

Special thanks to theHIV+ volunteers

Juila Sung MDCarolina Garrido Pavon, PhDNatalia Soriano, PhD

Nancie Archin, PhDNoelle DahlRosalie BatesonBrigette Allard

Joann Kuruc, MSN RNAmanda CrooksCynthia Gay, MD, MPH

Children’s National Medical Center Catherine Bollard, MDSharon Lam