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.