Laboratory ofExperim ental VirologyLaboratory ofExperim ental Virology

Level of HIV-1 unspliced RNA in PBMC: a longitudinal marker of infection progression

in untreated individuals and a predictive marker of virological rebound

in patients on ART with undetectable plasma viremia

Alexander Pasternak

Laboratory of Experimental VirologyDepartment of Medical Microbiology

Academic Medical CenterUniversity of Amsterdam

Amsterdam, The Netherlands

XVIII International AIDS Conference, July 22, 2010

gag

pol env

tat

rev

5’LTR nefvif

vpr

vpu 3’LTR

prDNA, usRNA

msRNA

Development of sensitive seminested real-time PCR methods

PCR I (conventional, 15 c)

PCR I (conventional, 15 c)

PCR II (real-time, 40-45 c)

PCR II (real-time, 40-45 c)

usRNA, prDNA msRNA

0,1

1

10

0 5 10 15 20 25 30 35 40 45 50

amplification cycle

delta

Rn

2.5*10(6)sn

5*10(5)sn

5*10(4)sn

5*10(3)sn

5*10(2)sn

1*10(2)sn

2*10(1)sn

4*10(0)sn

5*10(6)ss

5*10(5)ss

5*10(4)ss

5*10(3)ss

5*10(2)ss

1*10(2)ss

0,1

1

10

0 5 10 15 20 25 30 35 40 45

amplification cycle

delta

Rn

2.5*10(6)sn

5*10(5)sn

5*10(4)sn

5*10(3)sn

5*10(2)sn

1*10(2)sn

2*10(1)sn

4*10(0)sn

5*10(6)ss

5*10(5)ss

5*10(4)ss

5*10(3)ss

5*10(2)ss

1*10(2)ss

0,1

1

10

0 5 10 15 20 25 30 35 40 45 50

amplification cycle

delta

Rn

2.5*10(6)sn

5*10(5)sn

5*10(4)sn

5*10(3)sn

5*10(2)sn

1*10(2)sn

2*10(1)sn

4*10(0)sn

5*10(6)ss

5*10(5)ss

5*10(4)ss

5*10(3)ss

5*10(2)ss

1*10(2)ss

Proviral DNA

Pre-amplification results in 3-4 log stronger input signal

Unspliced RNA

Multiply spliced RNA

3.5 log 3.75 log

4.25 log

Seminested real-time PCR

Single-step real-time PCR

Sensitivity is increased 1.5 log without losing the linearity

pd

R2 = 0.992

R2 = 0.985

0

1

2

3

4

5

6

7

0 10 20 30 40 50

Ct

log

stan

dard

cop

y nu

mbe

r

new

old

Linear (new)

Linear (old)

Proviral DNA Unspliced RNA

Seminested real-time PCR

Single-step real-time PCR

Multiply spliced RNA

us

R2 = 0.987

R2 = 0.991

0

1

2

3

4

5

6

7

0 10 20 30 40 50

Ct

log

sta

nd

ard

co

py

nu

mb

er

new

old

Линейный (old)

Линейный (new)

ms

R2 = 0.991

R2 = 0.981

0

1

2

3

4

5

6

7

0 10 20 30 40 50

Ct

log

sta

nd

ard

co

py

nu

mb

er

new

old

Линейный (new)

Линейный (old)

1.5 log 1.5 log

1.5 log

HIV-1plasma load

50 copies/ml

start of cART

cART failures (n=15)

successes (n=11)

PBMC were isolated at baseline and at multiple time points during therapy

prDNA

usRNA

100%

100%

94%

94%

msRNA 83% 16%

Plasma RNA

0 1 2 3 40

2

4

6

time on cART, years

log

10 c

op

ies

/ ml

Proviral DNA

0 1 2 3 42

3

4

5

time on cART, years

log

10 c

op

ies

/ 1

06

PB

MC

Unspliced RNA

0 1 2 3 42

3

4

5

6

time on cART, years

log

10 c

op

ies

/ g

to

tal R

NA

CD4+ T cell count

0 1 2 3 40

200

400

600

800

1000

1200

time on cART, years

ce

lls /

mm

3 b

loo

d

Longitudinal trends of virological parametersand CD4+ count in patients on cART

P=0.001

P=0.002

P=0.008

Plasma RNA

0 1 2 3 40

2

4

6

time on cART, years

log

10 c

op

ies

/ ml

Proviral DNA

0 1 2 3 42

3

4

5

time on cART, years

log

10 c

op

ies

/ 1

06

PB

MC

Unspliced RNA

0 1 2 3 42

3

4

5

6

time on cART, years

log

10 c

op

ies

/ g

to

tal R

NA

CD4+ T cell count

0 1 2 3 40

200

400

600

800

1000

1200

time on cART, years

ce

lls /

mm

3 b

loo

d

Longitudinal trends of virological parametersand CD4+ count in patients on cART

P=0.001

P=0.002

P=0.008P=0.32

P=0.006

P=0.005

Plasma RNA

0 1 2 3 40

2

4

6

time on cART, years

log

10 c

op

ies

/ m

lProviral DNA

0 1 2 3 4

2

3

4

5

time on cART, years

log

10 c

op

ies

/ 1

06

PB

MC

Unspliced RNA

0 1 2 3 42

3

4

5

6

time on cART, years

log

10 c

op

ies

/ g

to

tal R

NA

CD4+ T cell count

0 1 2 3 40

200

400

600

800

1000

1200

time on cART, years

ce

lls /

mm

3 b

loo

d

Levels of virological parameters and CD4+ count in patients on cART

future failures (n=15)

successes (n=11)

Pdiff = 0.56

Pdiff = 0.02

Pdiff = 0.73

Pdiff = 0.64

Plasma RNA

0 1 2 3 40

2

4

6

time on cART, years

log

10 c

op

ies

/ m

lProviral DNA

0 1 2 3 4

2

3

4

5

time on cART, years

log

10 c

op

ies

/ 1

06

PB

MC

Unspliced RNA

0 1 2 3 42

3

4

5

6

time on cART, years

log

10 c

op

ies

/ g

to

tal R

NA

CD4+ T cell count

0 1 2 3 40

200

400

600

800

1000

1200

time on cART, years

ce

lls /

mm

3 b

loo

d

Levels of virological parameters and CD4+ count in patients on cART

future failures (n=15)

successes (n=11)

Pdiff = 0.24

Pdiff = 0.15Pdiff = 0.0015

2.5 3.0 3.5 4.0 4.5 5.0

0

20

40

60

80

100

usRNAMaximal usRNA level during cART,

log10 copies/µg total RNA

Ris

k o

f V

R, %

The risk of virological rebound according to the maximal usRNA value during cART

P=0.0033 (univariate)P=0.024 (multivariate)

future failures

successes

1

7

14

4

Correlations of virological parameters and CD4+ count

2 3 4 51

2

3

4

5

prDNA baseline

mea

n p

rDN

A c

AR

T rs = 0.74; P < 0.0001

3 4 5 62

3

4

5

usRNA baseline

mea

n u

sRN

A c

AR

T rs = 0.16; P = 0.45

0 200 400 600 8002

3

4

5

CD4 count baseline

mea

n u

sRN

A c

AR

T rs = -0.65; P = 0.0003

2 3 4 50

500

1000

1500

mean usRNA cART

mea

n C

D4

cou

nt

cAR

T rs = -0.56; P = 0.003

Conclusions (I)

Levels of usRNA in PBMC in patients on cART with undetectable plasma viremia are strongly predictive of the subsequent outcome of therapy.

Levels of prDNA gradually diminished during the therapy period in both patient groups, whereas levels of usRNA dropped sharply after the start of therapy, but subsequently reached the plateau, with the difference between the usRNA levels of future cART failures and successes established early after the start of cART and persisting throughout the therapy period.

Levels of usRNA under cART strongly inversely correlated with baseline CD4+ count, but did not correlate with either baseline usRNA levels or prDNA levels under therapy.

The observation that patients with higher levels of usRNA in PBMC were more prone to failing cART might link higher cellular HIV-1 RNA load in these patients with virus replication under therapy, which resulted in selection of drug-resistance mutations.

How about untreated patients?

Longitudinal trends of plasma HIV-1 RNA are highly variable between patients

No reliable longitudinal virological markers of HIV-1 infection progression in the asymptomatic phase are known

=>

How about untreated patients?

To compare the dynamics of HIV-1 molecular markers in PBMC and plasma in untreated infection,we selected 10 untreated patients and measured HIV-1 usRNA, msRNA, and prDNA levels

at multiple time points during the asymptomatic phase (clinical latency)

0.0 0.5 1.0 1.5 2.02.5

3.0

3.5

4.0

4.5

5.0

350

400

450

500

550

600USPDMSplasmaCD4

time, years

HIV-

1 RN

A/DN

A

CD4+ counts

0 2 4 6 80

2

4

6

200

400

600

800

1000

1200

PDMSplasmaCD4

US

time, years

HIV

-1 R

NA/

DN

A

CD

4+ counts

Plasma RNA

usRNA prDNA msRNA CD4+

count

Proportionsignificant

trends

positive 0/10 6/10 4/10 1/8 0/10

negative 0/10 0/10 0/10 0/8 5/10

Regression slopes in the individual patients

Plasma RNA

usRNA prDNA msRNA CD4+

count

Longitudinal trends of the analyzed parameters

US PD MS

US/PD

US/MS PL

CD4-1.0

-0.5

0.0

0.5

1.0

-200

-100

0

100

200

slo

pe,

log

10/y

ear

slop

e CD

4, cells/mm

3/year

** ** ***ns ns ns

**

*

Longitudinal slopes

US PD MS

US/PD

US/MS PL

CD4-1.0

-0.5

0.0

0.5

1.0

1.5

-600

-400

-200

0

200

400

600

TW

C,

log

10

TW

C C

D4, cells/m

m3

** ** ***ns ** ns

**

*

Time-weighted changes from baseline

US PD MS

US/PD

US/MS PL

CD4-2

-1

0

1

2

-1000

-500

0

500

1000

chan

ge,

log

10

chan

ge C

D4, cells/m

m3

** ** **ns ns nsns

**

Changes from baseline

Longitudinal trends of the analyzed parameters:linear mixed model

Correlations between the virological parametersand CD4+ count

3.0 3.5 4.0 4.5 5.0 5.50

200

400

600

800

1000

1200

usRNA

CD

4 co

un

t

2.0 2.5 3.0 3.5 4.0 4.50

200

400

600

800

1000

1200

prDNA

CD

4 co

un

t

rs = -0.43 rs = -0.46

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.00

200

400

600

800

1000

1200

plasma RNA

CD

4 co

un

t

rs = 0.13

Conclusions (II)

Levels of HIV-1 RNA and DNA in PBMC increased during the asymptomatic phase of infection in untreated individuals with stable RNA levels in plasma.

Levels of usRNA increased faster than those of prDNA: an increase in viral replication rates in PBMC.

Levels of usRNA and prDNA, but not those of plasma RNA, inversely correlated with CD4+ count, which is itself a marker of infection progression in the asymptomatic phase.

Level of usRNA in PBMC (which was shown earlier to be a predictive marker of the response to cART) is also a longitudinal marker of infection progression in the asymptomatic phase of the untreated infection.

Acknowledgements

Karen AdemaMargreet BakkerMarion CornelissenJoke Brouwer Suzanne Jurriaans

Ben BerkhoutVladimir Lukashov

Medical Microbiology, Academic Medical Center of the University of Amsterdam, The Netherlands

Laboratory ofExperim ental VirologyLaboratory ofExperim ental Virology

Jan Prins

Internal Diseases – Infectious Diseases, Tropical Medicine and AIDS, AMC, UvA

This study was financially supported by Dutch AIDS Fonds, project nr. 2004045

The problem is simple…

HIV-1plasma load

detectionlimit

start of cART

virological rebound, drug-resistant viruses

The problem is simple…

HIV-1plasma load

detectionlimit

start of cART

virological failure, drug-resistant viruses

How about untreated patients?

De Wolf et al., AIDS, 1997

Henrard et al., JAMA, 1995

Geskus et al., Retrovirology, 2007 Korenromp et al., PLoS ONE, 2009

Longitudinal trends of plasma HIV-1 RNA are highly variable between patients

No reliable longitudinal virological markers of HIV-1 infection progression in the asymptomatic phase are known

=>

Correlations between the virological parameters

3.0 3.5 4.0 4.5 5.0 5.52.0

2.5

3.0

3.5

4.0

4.5

usRNA

prD

NA

3.0 3.5 4.0 4.5 5.0 5.51.5

2.0

2.5

3.0

3.5

4.0

4.5

usRNA

msR

NA

3.0 3.5 4.0 4.5 5.0 5.52.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

usRNA

pla

sma

RN

A

rs = 0.69 rs = 0.46

rs = 0.42

Discussion (I)

Differences in longitudinal trends between prDNA and usRNA can be explained by the fact that prDNA levels reflect the size of the proviral archive, whereas levels of usRNA reflect the amounts of productively infected cells at every moment in time.

The observed difference in the usRNA levels between failures and successes may be shaped by the difference in the relative amounts of latently infected resting CD4+ T cells that become reactivated to the productively infected phenotype and escape immunological clearance.

Differences in longitudinal trends between prDNA and usRNA can be explained by the fact that prDNA levels reflect the size of the proviral archive, whereas levels of usRNA reflect the amounts of productively infected cells at every moment in time.

The relative amount of productively infected cells at any moment during cART (reflected in the usRNA level) might be defined, to a large extent, by the immunological state of the patient before therapy (reflected in the CD4+ count), and not simply by the size of viral reservoir (reflected in the levels of prDNA).

The observed difference in the usRNA levels between failures and successes may be shaped by the difference in the relative amounts of latently infected resting CD4+ T cells that become reactivated to the productively infected phenotype and escape immunological clearance.

The observation that patients with higher levels of usRNA in PBMC were more prone to failing cART might link higher cellular HIV-1 RNA load in these patients with virus replication under therapy, which resulted in selection of drug-resistance mutations.

The relative amount of productively infected cells at any moment during cART (reflected in the usRNA level) might be defined, to a large extent, by the immunological state of the patient before therapy (reflected in the CD4+ count), and not simply by the size of viral reservoir (reflected in the levels of prDNA).

Discussion (II)

So why is the increase in viral replication in PBMC is not (or, not significantly) paralleled by an increase in plasma viremia?

Plasma HIV-1 load reflects the balance between the virus production and clearance, whereas the cellular HIV-1 load would reflect the virus production only. It is possible that the ability of the immune system to clear free virus is compromised during the asymptomatic phase to a lesser extent than the ability to clear HIV-infected cells.

During this phase of infection, the weakening immune system may still possess a capacity to adjust the virus clearance rates to the increased virus production; when this capacity is exhausted, the uncontrolled virus replication rapidly leads to the onset of AIDS.

Alternatively, because most HIV-1 replication takes place in the lymphoid tissue, the virus produced by the infected PBMC may not substantially contribute to the total amount of free virus found in plasma.