HIV Cure Research Training Curriculum Scientific Leads Janet Siliciano PhD and Robert Siliciano MDPhD Johns Hopkins School of Medicine Community Leads Jeffrey Taylor CARE Nasra ID: 356433
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Slide1
Measuring the latent HIV Reservoir
HIV Cure Research Training Curriculum
Scientific Leads
: Janet Siliciano
, PhD and
Robert Siliciano,
MDPhD
,
Johns Hopkins School of Medicine
Community Leads
: Jeffrey Taylor, CARE;
Nasra
Aidarus
, AVAC
Module Contributors
: Jessica
Handibode
, AVAC and Karine Dubé, CARE
The HIV CURE research training curriculum is a collaborative project aimed at making HIV cure research science accessible to the community and the HIV research field. Slide2
Session Goals
Know what the latent reservoir is
Understand why targeting the reservoir is critical to achieving a cure
Name strategies to quantify the latent reservoirSlide3
What is viral latency?
Virus is present but not
active (not producing
HIV) in a cell
Virus is able to persist by integrating its genome into the host cell DNA
It remains “hidden” from immune responses
Reservoirs are cells where HIV is able to persist in the latent phase
Even while on antiretroviral therapySlide4
C
ell
D
eath
R
esting
S
tate
HIV persistenceSlide5
Naive
Ag
†
†
†
†
†
†
Memory
Establishment of immunologic memorySlide6
Naive
Ag
†
†
†
†
†
†
Ag
†
†
†
†
†
†
Memory
Establishment of immunologic memorySlide7
HIV infection of activated and resting CD4
+
T cells
†
Naive
Ag
†
HIV
Ag
HIV
HIV
MemorySlide8
Establishment of the latent reservoir in resting CD4+
T cells
Naive
Memory
Ag
†
†
†
†
†
†
HIVSlide9
What is the reservoir?
Latently infected cells
Cell type in which replication competent virus persists on the time scale of years in people on suppressive HAART
No known extracellular
markers associated with latency
The reservoir is established very early in infection but the exact timing is unknown
Palmer S. 2014. HIV Cure 101: Challenges in identifying and targeting the HIV reservoir. AIDS 2014 20
th
International AIDS Conference. Slide10
Viral latency and cure
Antiretroviral therapy can manage HIV infection and reduce viral load to undetectable levels
Despite undetectable viral load, the latent reservoirs still remain
Can be reactivated to produce HIV
ART prevents reinfection but is unable to target the reservoir.
Being off ART results in viral rebound, likely from reactivation of reservoir
Needs to be taken for lifeSlide11
Viral latency and cure
Latency is established within
cells infected before ART and can not be eliminated by ART
therapySlide12
Where are the reservoirs?
Cellular reservoirs
are widely dispersed throughout the body and can be in:
brain
lymphoid tissue
bone marrow
genital tract
Palmer S. 2014. HIV Cure 101: Challenges in identifying and targeting the HIV reservoir. AIDS 2014 20
th
International AIDS Conference. Slide13
Size of the reservoir
The size of the reservoir varies
The range can depend on several factors including timing
Timing of ART initiation – earlier initiation is associated with smaller reservoirsSlide14
Measuring the reservoir: why?
Essential to detect & quantify reservoir to evaluate if a cure has been achieved or to determine whether an intervention has reduced the latent reservoir
Need to be able to measure success of therapeutic agents charged with eradicationSlide15
Measuring the reservoir: how?
Currently the quantitative viral outgrowth assay (QVOA) is the gold standard used to measure the size of the latent reservoir
Common assays include:
PCR-based assays
Quantitative PCR (
qPCR
)
Reverse transcription PCR (
rtPCR)TILDA assay
Quantitative Viral Outgrowth Assay (QVOA) Gold StandardSlide16
Measuring proviral DNA: PCR
PCR
-based assays
detect viral DNA and are
commonly used in
labs
G
rossly
overestimate the size of the reservoir because they cannot distinguish defective vs. intact provirusMost of the proviruses are defectiveSlide17
Measuring proviral DNA: PCR
Quantitative PCR (
qPCR
)
measures the amplification of DNA using fluorescence
Fluorescence is proportional to the amount of PCR product
fluorescent
reporter
q
uencher
dye
probe
(can bind to target nucleotides)
Beacon.
When reporter and quencher are close, quencher absorbs fluorescenceSlide18
Measuring proviral DNA: PCR
Quantitative PCR (
qPCR
)
measures the amplification of DNA using fluorescence
Fluorescence is proportional to the amount of PCR product
p
rimer 1
p
rimer 2
Target PCR product
fluorescent
reporter
q
uencher
dye
probe
(can bind to target nucleotides)
Beacon.
When reporter and quencher are close, quencher absorbs fluorescenceSlide19
Measuring proviral DNA: PCR
Quantitative PCR (
qPCR
)
measures the amplification of DNA using fluorescence
Fluorescence is proportional to the amount of PCR product
Product detected by beacon.
Fluoresces once bound to target and separated from quencherSlide20
Measuring proviral DNA: PCR
qPCR
can
be used to
measure:
T
otal
& integrated HIV-1 DNA2. Two
long terminal repeat (LTR) circles
If can be detected in suppressed individuals, might
be due to ongoing, low level replication Not entirely clear if this is a reliable
markerSlide21
Measuring RNA:rt PCR
PCR only works on DNA
Reverse transcription PCR (
rtPCR
) used to measure free virus and virus gene expression.
RNA (from virus) is reverse transcribed into cDNA
The standard viral load assay is an
rtPCR
assay that detects viral RNA in virus particles.A more sensitive form of this assay can detect virus particles even in pateints with and “undetectable viral load”. This is the single copy assay for residual viremia (SCA assay)Slide22
Measuring HIV RNA Induction: TILDA
T
at/Rev
I
nduced
L
imiting
D
ilution AssayTILDA can be used as a screening assay to measure induction of HIV RNA in cells TILDA would yield a reservoir size in between VoA and DNA
Detects induction of latent proviruses but some may be defective
Chomont
N 2014 at Towards and HIV Cure Symposium, IASSlide23
Collect 10 to 20 mL of blood
Apply blood to
Ficoll
gradient centrifugation
TILDASlide24
Collect 10 to 20 mL of blood
Apply blood to
Ficoll
gradient centrifugation
Isolate CD4+ T cells from PBMC layer
TILDA
Ficoll
Blood
sample
PBMCs
Plasma
Ficoll
RBCs
c
entrifugeSlide25
TILDA
Split
isolated CD4+ T cells into two samples
Distribute both samples in limiting dilutions
Plate 1
Plate 2Slide26
TILDA
Add PMA and
ionomycin
cocktail to Plate 2
Used to stimulate CD4+ cells
7. Perform nested PCR on both plates
Plate 1
Plate 2
with
PMA and
ionomycin
Nested PCRSlide27
TILDA
Plate 1
Plate 2
+
PMA and
ionomycin
Nested PCRSlide28
TILDA
Results from Plate 1
Frequency of cells with
msHIV
RNA
(baseline)
Results from Plate 2
(stimulated with PMA +
ionomycin
)
Frequency of cells with inducible
msHIV
RNASlide29
Measuring the reservoir: VOA
V
iral
O
utgrowth
A
ssay
measures replication-competent HIVProvides a definitive minimal estimate of reservoir sizeOverview of process:Resting CD4+ T cells are activatedResting cells do not produce virus without
stimulationA
ctivation reverses latency
Virus is expanded in cells from uninfected donorsAdded at two different time points
Assay is assessed by ELISA for p24 (viral protein)
Ho, Cell 2013Slide30
Quantitative viral outgrowth assay
200 ml blood
Purified resting
CD4
+
T cells
Adapted from
Finzi
et al.,
Science, 1997
Blood is drawn and resting CD4+ T cells are purifiedSlide31
Quantitative viral outgrowth assay
200 ml blood
Cells are plated in dilution
p
urified resting
CD4
+
T cells
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide32
Quantitative viral outgrowth assay
200 ml blood
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
Cells are plated in dilution
1/1,000,000
p
urified resting
CD4
+
T cells
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide33
Quantitative viral outgrowth assay
200 ml blood
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
Cells are plated in dilution
1/1,000,000
p
urified resting
CD4
+
T cells
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide34
Quantitative viral outgrowth assay
Resting CD4 T cells are
activated using PHA.
Since resting
cells do not produce virus without
stimulation, PHA is used to reverse latency.
200 ml blood
p
urified resting
CD4
+
T cells
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
r
eactivation with PHA
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide35
Quantitative viral outgrowth assay
Latently infected cells can then then produce virus which is expanded
by add
CD4+
T cells
from
HIV negative
donors
200 ml blood
p
urified resting
CD4
+
T cells
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
v
irus amplification
Add CD4+ from HIV neg. donor
r
eactivation with PHA
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide36
Quantitative viral outgrowth assay
After two weeks, add more HIV negative CD4+ T-cells
200 ml blood
p
urified resting
CD4
+
T cells
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
v
irus amplification
Add CD4+ from HIV neg. donor
r
eactivation with PHA
Add CD4+ from HIV neg. donor
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide37
Quantitative viral outgrowth assay
HIVp24
Ag
200 ml blood
p
urified resting
CD4
+
T cells
Negative
control
1.6x10
2
5x10
6
10
6
2x10
5
4x10
4
8x10
3
Can now grow out from single latently infected cell enough virus to detect with an ELISA
v
irus amplification
Add CD4+ from HIV neg. donor
r
eactivation with PHA
Add CD4+ from HIV neg. donor
v
irus amplification
p
atient on ART
Adapted from
Finzi
et al.,
Science, 1997Slide38
Technical challenges in measuring the reservoir
Latently infected resting CD4+ T cells are present at low frequency and therefore large blood samples are required to measure them.
There may be other reservoirs, but this is not yet established
Not entirely known how the reservoir is establishedSlide39
Size of the latent reservoir
VOA
Intact
HIV DNA
Scale=100/10
6
Ho et al, Cell, 2013Slide40
Size of the latent reservoir
VOA
Intact
HIV DN
A
Scale=100/10
6
Ho et al, Cell, 2013Slide41
Size of the latent reservoir
VOA
Intact
HIV DN
A
Scale=100/10
6
Ho et al, Cell, 2013Slide42
Can intact non-induced proviruses be induced?
Ho et al, Cell, 2013
Nina Hosmane
Resting CD4
+
T cellsSlide43
Can intact non-induced proviruses be induced?
Ho et al, Cell, 2013
Nina Hosmane
47%
53%
PHA+
allo
PBMC
+
-
Resting CD4
+
T cellsSlide44
Can intact non-induced proviruses be induced?
Ho et al, Cell, 2013
Nina Hosmane
47%
53%
PHA+
allo
PBMC
+
-
39%
61%
PHA+
allo
PBMC
+
-
Resting CD4
+
T cellsSlide45
Can intact non-induced proviruses be induced?
Ho et al, Cell, 2013
Nina Hosmane
47%
53%
PHA+
allo
PBMC
+
-
39%
61%
PHA+
allo
PBMC
+
-
39%
61%
PHA+
allo
PBMC
+
-
Resting CD4
+
T cellsSlide46
Can intact non-induced proviruses be induced?
Ho et al, Cell, 2013
Nina Hosmane
47%
53%
PHA+
allo
PBMC
+
-
39%
61%
PHA+
allo
PBMC
+
-
39%
61%
PHA+
allo
PBMC
+
-
Resting CD4
+
T cellsSlide47
Infected cell frequencies
Ho et al, Cell, 2013
Katie Bruner, Nina
Hosmane
Viral outgrowth
assay
Cells with intact
provirus
Cells with HIV
DNA
Scale=1/10
6
450/10
6
15/10
6
1/10
6Slide48
Model for time to rebound
Hill et al, PNAS, 2014Slide49
What may HIV cure look like
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100
Plasma
HIV
RNA
(
copies/ml
)Slide50
What may HIV cure look like
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100
Plasma
HIV
RNA
(
copies/ml
)Slide51
What may HIV cure look like
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100
Plasma
HIV
RNA
(
copies/ml
)
cARTSlide52
What may HIV cure look like
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100
Plasma
HIV
RNA
(
copies/ml
)
cART
Therapeutic vaccination
cLRAsSlide53
What may HIV cure look like
Time Post Infection
(weeks) (years)
1,000,000
100,000
10,000
1000
100
Plasma
HIV
RNA
(
copies/ml
)
cART
Therapeutic vaccination
cLRAsSlide54
Global challenges in measuring the reservoir
Current assays are not available in resource limited settings.
They require cold-chain logistics, expensive machinery and are time consuming
Low and middle-income nations lack capacity and infrastructure to execute complex assays
Large barrier in scale-up and reproducibility internationally Slide55
Conclusions
Eliminating the reservoir is critical in order to achieve a functional or sterilizing HIV cure
Quantifying
the reservoir is still a challenge
Methods to precisely quantify the reservoir are being optimized
Need
for high-throughput, sensitive and valid assays for
reservoirSlide56
Module collaborators