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MEASURING THE LATENT HIV RESERVOIR
HIV Cure Research Training Curriculum The HIV CURE training curriculum is a collaborative project aimed at making HIV cure research science accessible to the community and the HIV research field.
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 reservoir
What is viral latency?• Virus is present but not active (not producing
HIV) in a cell
• Virus is able to persist by integrating it’s 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 therapy
What is the reservoir?• Latently infected cells• No universally accepted of
definition• No consensus on cellular
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 20th International AIDS Conference.
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 life
Viral latency and cure
Latency is established within cells infected before ART and can not be
eliminated by ART therapy
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 20th International AIDS Conference.
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 hypothesized to be associated with smaller reservoirs
Measuring the reservoir: why?• Essential to detect & quantify reservoir to
evaluate if a cure has been achieved• Need to be able to measure success of therapeutic
agents charged with eradication
• Size and location of reservoir may inform which intervention would be most suitable for use
Measuring the reservoir: how?• Currently there is no gold standard method
to measure the size of reservoir
Common assays include:1. PCR-based assays
a. Quantitative PCR (qPCR)b. Reverse transcription PCR (rtPCR)
2. TILDA assay3. Viral Outgrowth Assay (VOA)
Measuring the reservoir: PCR• PCR-based assays are reliable and commonly
used in labs
• May overestimate the size of the reservoir because can not distinguish defective vs. intact provirus
Measuring the reservoir: PCR• Quantitative PCR (qPCR) measures the
amplification of DNA using fluorescence• Fluorescence is proportional to amount of PCR product
primer 1
primer 2Target PCR product
fluorescent
reporter
quench
er dye
probe(can bind to target
nucleotides)
Beacon.When reporter and quencher are close, quencher absorbs fluorescence
Product detected by beacon. Fluoresces once bound to target and separated from quencher
Measuring the reservoir: PCRqPCR can be used to measure:
1. total & integrated HIV-1 DNA
2. Cell associated RNA (caRNA) Marker for frequency of latency Indicator of residual virus expression
3. 2 long terminal repeat (LTR) circles• Short-lived*• If can be detected in suppressed individuals,
must be due to ongoing, low level replication • Not entirely clear if this is a reliable marker
Measuring the reservoir: PCR• Reverse transcription PCR (rtPCR) used to
measure RNA expression• RNA (from virus) is cloned in DNA (complement DNA)
• The basis of rtPCR can be used in measuring singly copy RNA (SCA assay)
Measuring the reservoir: TILDA• Tat/Rev Induced Limiting Dilution Assay• TILDA gives a reservoir size in between VoA
and DNA
Chomont, 2014 at Towards and HIV Cure symposium, IAS
1. Collect 10 to 20mL of blood
2. Apply blood to Ficoll gradient centrifugation
3. Isolate CD4+ T cells from PBMC layer
Measuring the reservoir: TILDA
Ficoll
Blood sample
PBMCsPlasma
Ficoll
RBCs
centrifuge
Measuring the reservoir: TILDA
4. Split isolated CD4 T cells into two samples
5. Distribute both samples in limiting dilutions
Plate 1 Plate 2
Measuring the reservoir: TILDA6. Add PMA and ionomycin cocktail
to Plate 2i. Used to stimulate CD4 cells
7. Perform nested PCR on both plates
Plate 1
Plate 2with
PMA and ionomycin
Nested PCR
Measuring the reservoir: TILDA
Plate 1
Plate 2+
PMA and ionomycin
Nested PCR
Results from Plate 1Frequency of cells with msHIV RNA(baseline)
Results from Plate 2 (stimulated with PMA + ionomycin)
Frequency of cells with inducible msHIV RNA
Measuring the reservoir: VOA• Viral Outgrowth Assay measures replication-
competent HIV
• May largely underestimate the size of the reservoir• Also may not be suited for use in clinical trials
• Overview of process:1. Resting CD4 T cells are activated
a. Resting cells do not produce virus without stimulationb. activation reverses latency
2. Virus is expanded from uninfected donorsa. Added at two different time points
3. Assay is assessed by ELISA for p24 (viral protein)Ho, Cell 2013
Quantitative viral outgrowth assay200 ml blood
Purified restingCD4+ T cells
Adapted from Finzi et al., Science, 1997
Blood is drawn and resting CD4 T cells are purified
Quantitative viral outgrowth assay200 ml blood
Negative
contro
l
1.6x102
5x106
106
2x105
4x104
8x103
Cells are plated in dilution
1/1,000,000PURIFIED RESTING
CD4+ T CELLS
PATIENT ON ART
Adapted from Finzi et al., Science, 1997
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
PURIFIED RESTING
CD4+ T CELLS
Negative
contro
l
1.6x102
5x106
106
2x105
4x104
8x103
REACTIVATION WITH PHA
PATIENT ON ART
Adapted from Finzi et al., Science, 1997
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
PURIFIED RESTING
CD4+ T CELLS
Negative
contro
l
1.6x102
5x106
106
2x105
4x104
8x103
VIRUS AMPLIFICATION
ADD CD4+ FROM HIV NEG. DONOR
REACTIVATION WITH PHA
PATIENT ON ART
Adapted from Finzi et al., Science, 1997
Quantitative viral outgrowth assay
After two weeks, add more HIV negative CD4+ T-cells
200 ml blood
PURIFIED RESTING
CD4+ T CELLS
Negative
contro
l
1.6x102
5x106
106
2x105
4x104
8x103
VIRUS AMPLIFICATION
ADD CD4+ FROM HIV NEG. DONOR
REACTIVATION WITH PHA
ADD CD4+ FROM HIV NEG. DONOR
PATIENT ON ART
Adapted from Finzi et al., Science, 1997
Quantitative viral outgrowth assay
HIVp24Ag
200 ml blood
PURIFIED RESTING
CD4+ T CELLS
Negative
contro
l
1.6x102
5x106
106
2x105
4x104
8x103
Can now grow out from single latently infected cell to detect HIV
with an ELISA
VIRUS AMPLIFICATION
ADD CD4+ FROM HIV NEG. DONOR
REACTIVATION WITH PHA
ADD CD4+ FROM HIV NEG. DONOR
VIRUS AMPLIFICATION
PATIENT ON ART
Adapted from Finzi et al., Science, 1997
Technical challenges in measuring the reservoir• The reservoir is highly dispersed and not consistent
between individuals
• Not entirely known how the reservoir is established
• Need to identify reservoir sources and site(s) of viral rebound
• Mechanism for viral persistence has not been elucidated• Several models and theories have been proposed
Measuring the size of the reservoir
HIV Antigen (protein)detector
The “Real”Reservoir
HIV DNA
VOAgrowing virus
Limitations of assay types in
determining “real”
reservoir size
Global challenges in measuring the reservoir• Available assays are not reproducible
internationally
• They require cold-chain logistics, expensive machinery and time consuming
• Low and middle-income nations lack capacity and infrastructure to execute complex assays
• Large barrier in scale-up and reproducibility internationally
Conclusions• Eliminating the reservoir is critical in order
to achieve a sterilizing HIV cure
• Identifying & 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
reservoir