Where does HIV hide?Latent infection and cellular reservoirs

Nicolas ChomontProfessionnal development workshop, July 21, Vienna

Limit of detection

Antiretroviral drugs (HAART) are capable of suppressing HIV, even to

undetectable levels

Current Anti-HIV Drugs do not Eradicate HIV

Circ

ulat

ing

viru

s

Time

START STOP

HAART

However, the virus rebounds after

cessation of therapy

HIV hides in reservoir that are not sensitive to current therapies

HIV infection is characterized by high levels of circulating

viruses in the blood

How HIV persists?

1995: Resting CD4 T cells harbouring integrated provirus are present in HIV-infected individuals (TW Chun, Nat. Med).

1997: These cells persist in patients receiving suppressive HAART (D Finzi, Science ; TW Chun, PNAS).

1999: The half life of this reservoir is extremely long (D Finzi Nat. Med) but can be reduced by initiating HAART in acute infection (L Zhang, NEJM).

2005: Ongoing viral replication occurs in subjects on suppressive HAART and contributes to HIV persistence (TW Chun, JCI ; N Tobin, J Virol).

Now: HAART intensification studies (J Dinoso, PNAS ; D McMahon, CID; M Buzon, Nat Med)

Persistence of latently infected cells

Ongoing viral replication at low levels

How HIV persists?

How the HIV reservoir is established?

Ag Contraction

HIV

HIV

Suha Saleh, Blood, 2007Una O’Doherty, J Virol 2009

CCL19

Where HIV Persists During Antiviral Therapy?

• At the anatomical level: Potential “hiding places” for HIV:• Brain• Lymph nodes• Peripheral blood• Gut• Bone marrow

• At the cellular level: A small pool of cells harbor viral DNA integrated within the genome of the host. The frequency of these “reservoir cells” is very low (less than 1 in a million)

HIV can hide “dormant” for the lifetime of the cell

10

100

1000

10000

100000

Total HIV DNA

Proviral HIV DNA

2-LTR circles

HAARTViral loadWeeks

OFF3.107

-1

OFF1.105

-1

OFF3.104

-1

HIV

DN

A in

106

CD

4 T

ce l

ls

OFF5.105

-0.5

ON<50+90

ON<50+22

ON<50+52

ON<50+3

ON<50+14

ON<50+71

ON<50+11

ON<50+24

During HAART, HIV persists as an integrated provirus

How HIV Persists During Antiviral Therapy?

Several cellular reservoirs have been described in vivo (CD4+, CD8+, CD3+CD4-CD8-, NK cells, Monocytes, Dendritic cells…)

3 major caveats:1. Insufficient purity (results in false positive populations due to contamination

by CD4 T cells)2. Treatment duration too short (cellular reservoirs are different in viremic and

HAART patients) 3. Sensitivity of the assay (results in false negative populations)

HIV Reservoir localization

CD3 PE-Cy7

CD

14 F

ITC

CD4 APC

CD

8 P

erC

P C

y5.5

Strategy: cell sorting + quantification of HIV-1 DNA

HIV Reservoir Localization

CD3

CD

14

CD4

CD

8

Sorted CD4 T cells

Sorted CD8 T cells

Sorted DN T cells

Sorted Monocytes

HIV Reservoir Localization

Quantification of integrated HIV DNA by ultrasensitive real time PCR

CD4+ CD8+ DN Monocytes PBMC1

10

100

1000

10000

HIV

DN

A c

opie

s pe

r 10

6 c

ells

HIV integrated DNA is rarely detected in non CD4 T cells from HAART patients

HIV Integrated DNA in Sorted Cells

Con

trib

utio

n to

the

poo

l of

HIV

infe

cted

cel

ls

CD4+ T cells constitute more than 95% of the HIV-1 reservoirNON exhaustive approach: macrophages, NK, DC…?

CD4+ CD8+ DN Monocytes0

25

50

75

100

Contribution of cellular subsets to the HIV reservoir

HIV Reservoir Size

HIV Reservoir, decay?

Longitudinal measurements of the reservoir size indicate that this reservoir is extremely stable (half-life = 39-44 months in subjects who have initiated HAART during chronic infection).

Time to eliminate:105 cells: 54.8 years106 cells: 65.7 years107 cells: 76.7 years

0

200000

400000

600000

800000

1000000

1200000

0 500 1000

Tota

l num

ber

o f

rese

rvo i

r ce

lls

Linear scale

Time (months)

1

10

100

1000

10000

100000

1000000

0 200 400 600 800 1000

Tota

l num

ber

o f

rese

rvo i

r ce

lls

Log scale

Time (months)

Factors impacting on the Reservoir Size

1

10

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10000

Duration of exposure to HIV> 1 year< 1 year

Inte

grat

ed H

IV D

NA

cop

ies

per

106 C

D4

T c

ells

p < 0.0001

CD4/CD8 ratio> 1< 1

p < 0.0001

1

10

100

1000

10000

Inte

grat

ed H

IV D

NA

cop

ies

per

106 C

D4

T c

ells

0 2 4

1

10

100

1000

10000

% Ki67+ CD4 T cells

= 0.44p = 0.01

Inte

grat

ed H

IV D

NA

cop

ies

per

106 C

D4

T c

ells

Duration of exposure to HIV, CD4/CD8 ratio and Ki67 expression levels predict the HIV reservoir size

The CD4 compartment is heterogeneous

CD3 FITC

SS

C

CD4 APC

CD

8 P

erC

P-C

y5.5

CD45RA APC-Cy7

SS

C

CD27 PE

CC

R7

PE

-Cy7

Naïve cells have not seen their cognate antigen

Central memory cells: Long lived

Effector memory cells: Immediate effector functions

Transitionnal memory cells: Slowly proliferate

Terminally differentiated cells

0

20

40

60

80

100

TN TTDTCM

CD45RACCR7CD27

+++

+--

-++

TTM TEM

--+

---

Contributions of CD4 T Cell Subsets to the HIV ReservoirC

ontr

ibut

ion

(%)

to th

e H

IV r

eser

voir

size

%T

CM c

ells

am

ong

HIV

+ c

ells

= 0.66p = 0.004

%T

TM

+E

M c

ells

am

ong

HIV

+ c

ells

= -0.64p = 0.006

CD4 count (cells/µl)

CD4 count (cells/µl)

200 700 12000

20

40

60

80

100

200 700 12000

20

40

60

80

100

= -0.70p = 0.002

= 0.75p = 0.0006

% Ki67+ CD4 T cells

% Ki67+ CD4 T cells

0 1 2 3 40

20

40

60

80

100

0 1 2 3 40

20

40

60

80

100

TCM contribution

TTM contribution

CD4 Counts/Ki67 Levels and Reservoir Localization

Inte

grat

ed H

IV D

NA

cop

ies

per

106 C

D4

T c

ells

10

100

1000

10000

0 400 800 1200

Days Ki67+ TTM cells (%)

= -0.88p = 0.007

0.01

0.1

1

0 2 4 6S

lop

e d

ecre

ase

(% d

ecre

ase

/da

y)

Stability of the HIV reservoir size in patients with proliferating TTM and high IL-7 levels

=> Homeostatic proliferation of reservoir cells contribute to HIV persistence.

Stability of the Reservoir in Patients with Proliferating TTM

0.01

0.1

1

0 5 10IL-7 (pg/ml)

= -0.76p = 0.037

At least three mechanisms could be implicated:• Ongoing viral replication at low levels and replenishment of the pool of infected cells (particularly in privileged anatomical reservoirs such as the central nervous system and the gut).

• T cell survival (TCM): Reservoir cells are memory T cells. These cells, which are generated after infection or vaccination, keep the memory of the immune system for decades.

• Proliferation (TTM): Reservoir cells, like other memory T cells, divide very slowly to maintain the memory of the immune system (A. Bosque and V. Planelles)

How HIV persists during antiviral therapy?

T cell survival

Proliferation

Viral replication

Can we reduce the HIV reservoir size?

Possible strategies to eliminate/control/reduce the reservoir:• Start HAART during acute infection (Hocqueloux, AIDS 2010)• Intensification of HAART• Reactivation of HIV replication from its latent reservoir (D. Margolis, A. Savarino)

• Interfering with the immunological mechanisms that contribute to HIV persistence:

HIV-induced cell death

Uninfected cells

Cytokines, chemical compounds…

T cell survival Proliferation

Antibodies, cytokines, gene therapy, chemotherapy

Conclusion

• In individuals on suppressive HAART, HIV persists primarily in CD4 T cells.

• The HIV reservoir is stable and 70 years of continuous suppressive HAART may be necessary to eradicate HIV.

• Early HAART initiation limits the size of the viral reservoir.

• Within the CD4 compartment, TCM (long lived) and TTM (slowly proliferating) constitute the 2 majors viral reservoirs.

• At least 3 mechanisms contribute to HIV persistence: - Ongoing viral replication (Gut, CNS etc…) - T cell survival - Homeostatic proliferation

Their relative contributions are unknown but are likely to differ between individuals

VGTI Florida Université de Montréal, CHUMINSERM U743Sandrina Da FonsecaClaire VandergeetenMohamed El Far Petronela AncutaLydie TrautmannFrancesco ProcopioBader Yassine-DiabGenevieve BoucherElias Haddad Rafick-Pierre Sékaly Royal Victoria Hospital, Mc Gill UniversityJean-Pierre Routy Mohamed-Rachid BoulasselGeorges GhattasVRC, NIAID, NIHDanny DouekJason BrenchleyBrenna Hill

Acknowledgements

The patients!!