1. 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.

2. 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

3. 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
Integration allows the viral genome to remain in the cell as it replicates (from generation to generation). Because of no active HIV production, the dormant state is able to evade host immune responses that require epitopes/antigens to initiate their cascade. This integration allows viral reproduction to initiate again later (likely when ART is interrupted).
Latency is also referred to as the “proviral” state.
Note difference between viral and clinical latency! Different terms.
Clinical latency is: virus is not dormant, replicating but host is not experiencing symptoms

4. 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
Latently infected cells can be resting CD4 cells or other cells that are infected with HIV
Established early in infection, usually before ART initiation –for this reason, there is additional benefit for early initiation of treatment. If treatment is started earlier,
Palmer S HIV Cure 101: Challenges in identifying and targeting the HIV reservoir. AIDS th International AIDS Conference.

5. 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
If only one antriretroviral drug is taken, HIV would become resistant (quickly) and the drug would stop working). Taking two or more ARVs is effective and successfully manages HIV infection. There are more than 20 approved drugs available. If prescribed regiment is adhered to and individuals are retained in care, viral load can be suppressed to undetectable levels.
“Undetectable” is a viral level that is too low to be detected my modern assays. This however does not mean that virus is not present.
The reservoir is a major barrier in eradication of HIV.
Antiretroviral therapy can control viral load and manage progression to AIDS by blocking production and spread of infection BUT is not able to target and eliminate already integrated, latent pools of virus.

6. Viral latency and cure
Latency is established within cells infected before ART and can not be eliminated by ART therapy
ART can keep reactivation of reservoirs as bay by blocking the reproduction process. But latent pools will always be present and can reactivate when treatment is interrupted. Cure options being assessed may include an ART component that will purge the reservoir after viral reactivation. But adherence to ART alone will not eliminate the reservoir and lead to a cure.

7. Where are the reservoirs?
Cellular reservoirs are widely dispersed throughout the body and can be in:
brain
lymphoid tissue
bone marrow
genital tract
Latently infected resting CD4+ T cells are well known and recognized. Other sources of reservoir are not well defined such as bone marrow or microglia in the brain (where ARVs can’t access because of the blood-brain barrier).
Due the complex mechanisms that dictate maintenance of the reservoir, the expression of these mechanisms vary depending on location and cell type. This lends to the diversity of the reservoir, one of the challenges of elimination.
Locations (other than blood) become more challenging to study because of the need to persistently & consistently sample before and after interventions to determine efficacy of clearance.
Palmer S HIV Cure 101: Challenges in identifying and targeting the HIV reservoir. AIDS th International AIDS Conference.

8. 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
Size of the reservoir varies based on time from infection to start of treatment and also on location (i.e. reservoir in GALT vs. brain).
A large piece of the puzzle (to be explored in later sections) is the ability to accurately quantify the actual size of the reservoir. Assays being employed for this purpose produce different estimates for size which contributes to the confusion/lack of clear understanding.
The thought with early ART initiation is that ARVs stop seeding of the reservoir. Recent data showing this comes from a clinical trial with Steve Deeks showing initiation during the “hyperacute” phase. Men enrolled in a PrEP study seroconverted after randomization but before the intervention (Truvada) and researchers were unaware of their status until follow up post PrEP administration. Results: men showed some sort of benefit immunologically particularly in terms of controlling virus and initiation during this hyperacute phase significantly decreased the size of their reservoir.
(TO KARINE & JESS: I am totally recalling this from memory from an amfAR meeting last year, can’t find any updated data?)

9. 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
Need to KNOW when cure is achieved. Ex. Boston Patients – did not realize that they had not achieved the cure until viral rebound was detected. Used several assays to quantify and search for residual virus and were unsuccessful.
The size of the reservoir may influence level of intensity of intervention whether a relatively more aggressive mechanism needs to be employed.

10. Measuring the reservoir: how?
Currently there is no gold standard method to measure the size of reservoir
Common assays include:
PCR-based assays
Quantitative PCR (qPCR)
Reverse transcription PCR (rtPCR)
TILDA assay
Viral Outgrowth Assay (VOA)

11. 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
PCR is a molecular technique that generates many (sometimes millions!) of copies of DNA. It helps to multiply DNA sequences to a large enough quantity to me measured. A PCR will amplify a sequence of DNA even if the actual protein (translated from the sequence) is defective. PCR results don’t distinguish between defective vs. intact virus (what we really want to measure)
Un-integrated DNA can be quantified by taking the difference between total HIV-DNA and (integrated DNA + 2-LTR DNA)

12. Measuring the reservoir: PCR
Quantitative PCR (qPCR) measures the amplification of DNA using fluorescence
Fluorescence is proportional to amount of PCR product
fluorescent reporter
quencher dye
probe
(can bind to target nucleotides)
Beacon.
When reporter and quencher are close, quencher absorbs fluorescence
qPCR uses the basis of PCR but is able to go one step further and determine the quantity of DNA in real time. In the example above, a sequence specific fluorescent probe is being used to detect the amplified product. In the beacon, the nucleotides are complementary to the target PCR product and bind when the product is present. This ensures that non-specific binding does not occur since fluorescence will only be achieved once the probe binds. Fluorescence occurs because during probe + product binding, the fluorescent dye is separated from the quencher. As you can see, in “closed” beacon form the dye and quencher are close together – the fluorescence being emitted in being “quenched” in this state. When they are separated (when the probe is open and binding), the dye is able to be emitted and detected through the assay machinery.
qPCR can be conducted without a fluorescent reporter probe method but instead using a dyes that bind any double stranded DNA amplified. This method is less expensive but specificity is sacrificed since all products (even non-targets) produced are detected.
Once reporter and quencher are separated, fluorescence can be detected.
Product detected by beacon. Fluoresces once bound to target and separated from quencher
primer 1
primer 2
Target PCR product

13. Measuring the reservoir: PCR
qPCR can be used to measure:
total & integrated HIV-1 DNA
Cell associated RNA (caRNA)
Marker for frequency of latency
Indicator of residual virus expression
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
qPCR can be used to measure integrated DNA specifically.
2. caRNA is a virological bimarker and is used as an indicator of virus replication and in determining the size of the reservoir. This is reviewed in the paper by Pasternak et al in Retrovirology 2013.
Side note: caHIV DNA is used for diagnosing infants 
3. 2-LTR circles is used to assess ongoing viral replication in patients on ART. Long terminal repeats are large nucleotide sequences that flank sequences of viral DNA. They are used for insertion of viral genetic material into hosts.
*the half life is debated since in vivo and in vitro data have not come to a consensus.

14. 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)
rtPCR uses the foundational basis of PCR but the start product is RNA that is converted into DNA (reverse transcribed) which is then amplified. rtPCR can be combined with real-time techniques for RT qPCR.
SCA assay overview: assay is able to detect RNA below the “undetectable” limit in current, FDA approved assays. Uses a plasma sample where the virion is recovered and digested (to remove protein primarily). RNA is precipitated and applied to an RT qPCR assay. (Hilldorfer et al 2012 in Curr HIV/AIDS Rep).

15. Measuring the reservoir: TILDA
Tat/Rev Induced Limiting Dilution Assay
TILDA gives a reservoir size in between VoA and DNA
Tat: HIV-associated protein. It is a regulatory protein that contributes to improving viral transcription.
Rev: HIV-associated protein. It is a regulatory that contributes to improving viral translation.
Chomont, 2014 at Towards and HIV Cure symposium, IAS

16. Measuring the reservoir: TILDA
Collect 10 to 20mL of blood
Apply blood to Ficoll gradient centrifugation
Isolate CD4+ T cells from PBMC layer
Ficoll
Blood
sample
PBMCs
Plasma
Ficoll
RBCs
centrifuge
Ficoll: ficoll is a density gradient that when applied to blood will allow for the separation and isolation of peripheral blood mononucleocytes (PBMC). Centrifugation (rapid spinning).

17. Measuring the reservoir: TILDA
Split isolated CD4 T cells into two samples
Distribute both samples in limiting dilutions
Limiting dilution: a process that aims to get a single monoclonal cell population from a polyclonal batch of cells. A limiting dilution is essentially a series of increasing dilutions (the dark to light in the figure represents high to low concentrations respectively). The circles represent single wells in a plate and each row is one dilution (higher dilution towards the bottom).
Plate 1
Plate 2

18. Measuring the reservoir: TILDA
Add PMA and ionomycin cocktail to Plate 2
Used to stimulate CD4 cells
Perform nested PCR on both plates
Plate 1
Nested PCR
Plate 2
with
PMA and
ionomycin

19. Measuring the reservoir: TILDA
Results from Plate 1
Frequency of cells with msHIV RNA
(baseline)
Plate 1
Nested PCR
Results from Plate 2
(stimulated with PMA + ionomycin)
Frequency of cells with inducible msHIV RNA
Plate 2 +
PMA and
ionomycin
msHIV RNA = multiply spliced HIV RNA

20. 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:
Resting CD4 T cells are activated
Resting cells do not produce virus without stimulation
activation reverses latency
Virus is expanded from uninfected donors
Added at two different time points
Assay is assessed by ELISA for p24 (viral protein)
Not suited for CT use because of cost and tie required. Needs two weeks of cell culture and PBMC from at least three HIV negative blood donors per assay.
Ho, Cell 2013

21. Quantitative viral outgrowth assay
200 ml blood
Purified resting
CD4+ T cells
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
Blood is drawn and resting CD4 T cells are purified
Adapted from Finzi et al., Science, 1997

22. Quantitative viral outgrowth assay
patient on ART
200 ml blood
Negative
control
1.6x102
5x106
106
2x105
4x104
8x103
1/1,000,000
purified resting
CD4+ T cells
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
Cells are plated in dilution
Adapted from Finzi et al., Science, 1997

23. Quantitative viral outgrowth assay
patient on ART
200 ml blood
Negative
control
1.6x102
5x106
106
2x105
4x104
8x103
purified resting
CD4+ T cells
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
Resting CD4 T cells are activated using PHA.
Since resting cells do not produce virus without stimulation, PHA is used to reverse latency.
reactivation with PHA
Adapted from Finzi et al., Science, 1997

24. Quantitative viral outgrowth assay
patient on ART
200 ml blood
Negative
control
1.6x102
5x106
106
2x105
4x104
8x103
purified resting
CD4+ T cells
Latently infected cells can then then produce virus which is expanded by add CD4+ T cells from HIV negative donors
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
Add CD4+ from HIV neg. donor
reactivation with PHA
virus amplification
Adapted from Finzi et al., Science, 1997

25. Quantitative viral outgrowth assay
patient on ART
200 ml blood
Negative
control
1.6x102
5x106
106
2x105
4x104
8x103
purified resting
CD4+ T cells
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
After two weeks, add more HIV negative CD4+ T-cells
Add CD4+ from HIV neg. donor
Add CD4+ from HIV neg. donor
reactivation with PHA
virus amplification
Adapted from Finzi et al., Science, 1997

26. Quantitative viral outgrowth assay
patient on ART
200 ml blood
Negative
control
1.6x102
5x106
106
2x105
4x104
8x103
purified resting
CD4+ T cells
Can now grow out from single latently infected cell to detect HIV with an ELISA
Resting CD4+ T cells are purified from large blood samples from patients on antiretoviral therapy. Cells are plated in limiting dilution and subjected to maximum activation with a mitogen that induce 100% of the resting CD4+ T cells to undergo blast transformation. Latently infected cells can then then produce virus which is expanded through coculture with CD4+ lymphoblasts from normal donors. After two weeks, you can grow out from a single latently infected cell enough virus to detect by Elisa assay. With this assay we showed these cells are present in everybody with HIV infection, but only at extremely low frequency. The frequency of cells about one in a million.
HIVp24 Ag
Add CD4+ from HIV neg. donor
Add CD4+ from HIV neg. donor
reactivation with PHA
virus amplification
virus amplification
Adapted from Finzi et al., Science, 1997

27. 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
As illustrated earlier, latent pools can be present in any combination of the sites listed (may be others unidentified). The location of reservoirs are not the same between different individuals.
When virus replicates from the latent pool after treatment interruption, unclear if where the rebound source is consistently, or if there is more than one.
Why and how viral reservoirs continue to be maintained is complex and not entirely known.

28. Measuring the size of the reservoir
HIV Antigen
(protein)
detector
The “Real”
Reservoir
HIV DNA
VOA
growing virus
Limitations of assay types in determining “real” reservoir size
Figure shows that PCR assays overestimate the reservoir while VOA may underestimate the size. Somewhere in-between these assays is where researchers theorize the size of the reservoir ACTUALLY is. Note: remember that the size of the reservoir does vary between individuals also.

29. 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
Need to have assays and interventions that can be applied to resource-poor settings that certainly experience the greatest burden of HIV/AIDS. Having equitable distribution of cure strategies should be a clear goal. Low income nations do not have the capacity currently to support difficult, complex, expensive assays and interventions. Particularly those that require storage, constant sources of electricity (for cold chain) and reliable transportation. These considerations need to be made when designing and developing cure strategies.

30. 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