Cell- and Tissue-based Measures of Viral Persistence Are Associated with

Immune Activation and PD-1-Expressing CD4+ T cells

H Hatano1, V Jain1, PW Hunt1, JN Martin1, TH Lee2, E Sinclair1, JM McCune1, F Hecht1, MP Busch1,2, SG Deeks1

1University of California, San Francisco, CA, USA2Blood Systems Research Institute, San Francisco, CA, USA

What are the determinants of HIV persistence?

• Determinants of HIV persistence during long-term HAART remain unknown, but may include:– Ongoing viral replication (Buzon, Nat Med 2010)

– Potency of HIV-specific responses• Mucosal HIV-specific T cell responses (Hatano, JID 2011)

Higher Levels of GALT HIV-specific CD8+ T Cells Are Associated with Lower Levels of Proviral DNA

rho = - 0.50, p = 0.03

CD4CD8

rho = - 0.42, p = 0.07

0 1 2

0

1

2

3

4

5

log10 Proviral DNA(per mil PBMC)

% G

ag-s

peci

ficC

D4+

T c

ells

(GA

LT)

0 1 2

0.0

0.5

1.0

1.5

log10 Proviral DNA(per mil PBMC)

% G

ag-s

peci

ficC

D8+

T c

ells

(GA

LT)

Hatano et al, JID 2011

What are the determinants of HIV persistence?

• Determinants of HIV persistence during long-term HAART remain unknown, but may include:– Persistent immune activation

• Immune activation levels remain elevated despite effective HAART-mediated viral suppression

– Negative regulators that reverse activated state (PD-1) (Chomont, Nat Med 2009)• PD-1high CD4+ T cells have high levels of proviral DNA• Triggering of PD-1 inhibits HIV transcription, and inhibiting the

PD-1/PD-L1 interaction increases HIV production• PD-1 expressing CD4+ T cells → Preferential reservoir for HIV

• Understanding the causes of persistent inflammation are important for preventing non-AIDS morbidity and for strategies towards cure

Study Objectives• To assess the relationship between measurements

of viral persistence and immune activation– Plasma RNA– Cell-associated RNA and proviral DNA– Tissue-associated RNA and proviral DNA

• To determine the relationship between treatment response and – T cell activation/dysfunction – Viral persistence

• To identify potential interventions to decrease HIV persistence

Methods• 190 HAART-suppressed subjects identified

from UCSF SCOPE/OPTIONS cohorts• Ultrasensitive plasma HIV RNA

– Modified Roche CAP/CTM v2.0 (LOD <5 copies/mL)• Cell-associated RNA• Proviral DNA• Immune activation (% CD4+ and CD8+ T cells)

– CD38+HLA-DR+, PD-1• Gut-associated lymphoid tissue (GALT)

samples were obtained from 14 subjects

Baseline Characteristics(n=190)

CD4 count (cells/mm3) 523

Age (years) 51

Gender (% male) 92%

Duration VL suppression (months) 31

Nadir CD4 count (cells/mm3) 113

No Association Between Low-Level Plasma RNA Levels and T Cell Activation

All p-values > 0.20

Modest Association Between Cell-Based Measures of Viral Persistence and T Cell Activationrho = 0.23, p = 0.014 rho = 0.16, p = 0.057

rho = 0.22, p = 0.008 rho = 0.14, p = 0.088

Highly Significant Association between Proviral DNA Levels and Frequency of PD-1

Expressing CD4+ T Cells

rho = 0.28, p = 0.0005

These observations are consistent with

PD-1 being a marker of latently infected CD4+ T

cells (Chomont et al, Nature Med

2009)

Strong Association Between Viral Persistence in GALT and T Cell Activation

What is the relationship between treatment response and…

-Viral Persistence? -T Cell Activation/Dysfunction?

Will treated subjects with a low CD4+ T cell count require unique curative strategies?

Plas

ma

HIV

RN

A(c

opie

s/m

L)

Low CD4 High CD40

50

100

150

Plasma RNA Levels Similar in Low and High CD4+ Groups

p = NS

Cell-Associated RNA and Proviral DNA Levels Are Higher in Low CD4+ Group

p = 0.008 p = 0.001

CD4+ T Cell Activation and PD-1 Expression are Higher in Low CD4+ Group

%C

D38

+HLA

-DR

+C

D4+

T c

ells

Low CD4 High CD40

5

10

15

20

25

p < 0.0001

%PD

1+ C

D4+

T c

ells

Low CD4 High CD40

20

40

60 p < 0.0001

%C

D38

+HLA

-DR

+PD

1+C

D4+

T c

ells

Low CD4 High CD40

5

10

15

p < 0.0001%

CD

38+H

LA-D

R+C

CR

5+PD

1+C

D4+

T c

ells

Low CD4 High CD40

2

4 p < 0.0001

Higher Frequencies of PD-1 Expressing TCM CD4+ T Cells in Low CD4+ Group

% C

D45

RA-

CC

R7+

PD1+

CD

4+ T

cel

ls

Low CD4 High CD40

5

10

15

20

p < 0.0001

% C

D45

RA-

CC

R7+

CD

38+H

LA-D

R+P

D1+

CD

4+ T

cel

ls

Low CD4 High CD40

2

4

6

p < 0.0001%

CD

45R

A-C

CR

7+C

D38

+HLA

-DR

+CC

R5+

PD1+

CD

4+ T

cel

ls

Low CD4 High CD40.0

0.5

1.0

1.5

p < 0.0001

Conclusions I.• No associations between ultrasensitive

plasma HIV RNA levels and immune activation

• Cell-based measurements of viral persistence were modestly but consistently associated with markers of immune activation/dysfunction and frequency of PD-1 expressing CD4+ T cells

• Stronger positive correlation between tissue-based measurements of viral persistence and immune activation

• Highly significant association between proviral DNA levels and frequency of PD-1 expressing CD4+ T Cells

• Phase I study of an anti-PD-1 monoclonal antibody aimed at clearing the latent reservoir is in development (ACTG 5301)

Potential Eradication Strategies

ACTG 5301 Study Schema• Study Design: Single arm, dose-finding study• Population:

– HIV-infected female and male subjects ≥ 18 years of age. Females of reproductive potential are excluded from the study.

– Screening CD4+ T cell count > 350 cells/mm3

– Plasma HV RNA < 75 copies/mL while taking HAART for previous 36 months

• Sample size: 40 (10 subjects in each dose cohort)• Study duration: 16 weeks• Intervention: Single IV dose of open-label MK3475

at dose of 0.1, 1, 3, or 10 mg/kg

• Treated patients with a low CD4+ T cell count had:– Higher cell-based measures of viral

persistence– Expansion of CD4+ T cells expressing PD-1

• Most consistently observed in the central memory compartment 

• Treated individuals with low CD4+ T cell counts may be more difficult to cure and/or will require unique interventions

Conclusions II.

Implications

• Understanding the causes of viral persistence and inflammation in the setting of HAART are necessary to develop new strategies towards cure

• Future studies of viral persistence should focus on cell- and tissue-based measurements of viral persistence, not on plasma RNA (Chun, JID 2008; Yukl, JID 2010;

Hatano, JID 2011)

AcknowledgementsUCSF/SFGH/PHP UCSF/SFGH/DEM FundingVivek Jain Elizabeth Sinclair NIAID K23AI075985Peter Hunt Joseph M. McCune Ma SomsoukJeffrey Martin VGTI Florida Frederick Hecht Nicolas ChomontSteven Deeks Rafick Sekaly

UCSF/SFVAMC Roche, Inc.Steven Yukl Tri DoJoseph Wong

UCSF/BSRITzong-Hae Lee Michael Busch