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.

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 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 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.

HIV persistence Cell Death Resting State Explain the figure: Cells infected with HIV are generally activated and the majority end in cell death – but not all of them. A minor population of infected cell reverts back to a resting state and can persist indefinitely without being detected by the immune system or antiretroviral therapy.

Establishment of immunologic memory Naive Ag † Memory These are latently infected resting CD4+ cells that arise as a consequence of the normal way in which immunologic memory is established. When a resting CD4+ cell encounters antigen, it undergoes blast transformation and divides, ultimately generated many activated effector cells with the same specificity. At the conclusion of the immune response, many of these activated cells die, but some survive and return to a resting state as long lived memory cells that allow future responses to the same antigen.

Establishment of immunologic memory Naive Ag † Memory These are latently infected resting CD4+ cells that arise as a consequence of the normal way in which immunologic memory is established. When a resting CD4+ cell encounters antigen, it undergoes blast transformation and divides, ultimately generated many activated effector cells with the same specificity. At the conclusion of the immune response, many of these activated cells die, but some survive and return to a resting state as long lived memory cells that allow future responses to the same antigen.

HIV infection of activated and resting CD4+ T cells † Naive Ag HIV Memory HIV replicates mainly in the activated cells. and these infected cells die quickly, hence the rapid decay rate a. The virus does not replicate well in resting T cells.

Establishment of the latent reservoir in resting CD4+ T cells Naive Memory Ag † HIV But on rare occasions activated T cells can become infected as they are returning to a resting state. This results in a stably integrated viral genome in a long lived memory T cell. And what is particularly interesting is that as the cell returns to a resting memory state, HIV gene expression is turned off

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 Latently infected cells can be resting CD4+ cells or other cells that are infected with HIV. They are 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, the HIV reservoir size is usually smaller. See early ART module for more details. 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 If only one antiretroviral 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.

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 at 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.

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 to HIV 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. 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 associated with smaller reservoirs The size of the HIV 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. Earlier treatment initiation is associated with a smaller HIV reservoir size. See early ART module for details.

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 eradication Need to be able to detect the presence of replication-competent provirus. For example, with the Boston Patients, scientists realized that they had not achieved the cure until viral rebound was detected.

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 Standard

Measuring proviral DNA: PCR PCR-based assays detect viral DNA and are commonly used in labs Grossly overestimate the size of the reservoir because they cannot distinguish defective vs. intact provirus Most of the proviruses are defective 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)

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 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.

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 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. primer 1 primer 2 Target PCR product

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 quencher 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.

Measuring proviral DNA: PCR qPCR can be used to measure: Total & integrated HIV-1 DNA 2. 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 marker qPCR can be used to measure integrated DNA specifically. 2. ca-RNA is a virological biomarker 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: ca-HIV 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.

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) 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).

Measuring HIV RNA Induction: TILDA Tat/Rev Induced Limiting Dilution Assay TILDA 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 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. TILDA: This assay should be used as a screening assay to measure HIV RNA induction after treatment with putative latency-reversing agents. It should not be used to measure the size of the replication-competent HIV reservoir. The TILDA measures RNA and a lot of it can contact defective HIV provirus. Chomont N 2014 at Towards and HIV Cure Symposium, IAS

TILDA Collect 10 to 20 mL of blood Apply blood to Ficoll gradient centrifugation Ficoll is a density gradient that when applied to blood will allow for the separation and isolation of peripheral blood mononucleocytes (PBMC). RBCs: Red blood cells. Centrifugation (rapid spinning).

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

TILDA Split isolated CD4+ T cells into two samples Distribute both samples in limiting dilutions Limiting dilution: 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

TILDA Add PMA and ionomycin cocktail to Plate 2  Used to stimulate CD4+ cells 7. Perform nested PCR on both plates Plate 1 Nested PCR Plate 2 with PMA and ionomycin

TILDA Plate 1 Nested PCR Plate 2 + PMA and ionomycin msHIV RNA = multiply spliced HIV RNA

(stimulated with PMA + ionomycin) TILDA Results from Plate 1 Frequency of cells with msHIV RNA (baseline) msHIV RNA = multiply spliced HIV RNA 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 Provides a definitive minimal estimate of reservoir size Overview of process: Resting CD4+ T cells are activated Resting cells do not produce virus without stimulation Activation reverses latency Virus is expanded in cells from uninfected donors Added at two different time points Assay is assessed by ELISA for p24 (viral protein) Needs two weeks of cell culture and PBMC from at least three HIV negative blood donors per assay Takes a long time Ho, Cell 2013

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 antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Blood is drawn and resting CD4+ T cells are purified Adapted from Finzi et al., Science, 1997

Quantitative viral outgrowth assay patient on ART 200 ml blood purified resting CD4+ T cells Resting CD4+ T cells are purified from large blood samples from patients on antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Cells are plated in dilution Adapted from Finzi et al., Science, 1997

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 antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Cells are plated in dilution Adapted from Finzi et al., Science, 1997

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 antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Cells are plated in dilution Adapted from Finzi et al., Science, 1997

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 antiretroviral 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 produce virus which is expanded through co-culture 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 is 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

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 antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Add CD4+ from HIV neg. donor reactivation with PHA virus amplification Adapted from Finzi et al., Science, 1997

Quantitative viral outgrowth assay patient on ART 200 ml blood Negative control 1.6x102 5x106 106 2x105 4x104 8x103 purified resting CD4+ T cells After two weeks, add more HIV negative CD4+ T-cells Resting CD4+ T cells are purified from large blood samples from patients on antiretroviral 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 produce virus which is expanded through co-culture 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 is about one in a million. Add CD4+ from HIV neg. donor Add CD4+ from HIV neg. donor reactivation with PHA virus amplification Adapted from Finzi et al., Science, 1997

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 enough virus to detect with an ELISA Resting CD4+ T cells are purified from large blood samples from patients on antiretroviral 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 produce virus which is expanded through co-culture 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 is 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

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 established 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 consistent, or if there is more than one. Why and how viral reservoirs continue to be maintained is complex and not entirely known.

Size of the latent reservoir HIV DNA Intact VOA Scale=100/106 Ho et al, Cell, 2013

Size of the latent reservoir HIV DNA Intact VOA Scale=100/106 Ho et al, Cell, 2013

Size of the latent reservoir HIV DNA Intact VOA Scale=100/106 Ho et al, Cell, 2013

Can intact non-induced proviruses be induced? Resting CD4+ T cells Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD4+ T cells PHA+ allo PBMC + - 47% 53% Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD4+ T cells PHA+ allo PBMC + - 47% 53% PHA+ allo PBMC + - 39% 61% Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD4+ T cells PHA+ allo PBMC + - 47% 53% PHA+ allo PBMC + - 39% 61% PHA+ allo PBMC + - 39% 61% Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD4+ T cells PHA+ allo PBMC + - 47% 53% PHA+ allo PBMC + - 39% 61% PHA+ allo PBMC + - 39% 61% Ho et al, Cell, 2013 Nina Hosmane

Infected cell frequencies 450/106 Cells with HIV DNA 15/106 1/106 Cells with intact provirus Viral outgrowth assay Ho et al, Cell, 2013 Katie Bruner, Nina Hosmane Scale=1/106

Model for time to rebound Hill et al, PNAS, 2014

What may HIV cure look like 1,000,000 100,000 10,000 Plasma HIV RNA (copies/ml) 1000 100 (weeks) (years) Time Post Infection

What may HIV cure look like 1,000,000 100,000 10,000 Plasma HIV RNA (copies/ml) 1000 100 (weeks) (years) Time Post Infection

What may HIV cure look like 1,000,000 100,000 cART 10,000 Plasma HIV RNA (copies/ml) 1000 100 (weeks) (years) Time Post Infection

What may HIV cure look like 1,000,000 Therapeutic vaccination 100,000 cART cLRAs 10,000 Plasma HIV RNA (copies/ml) 1000 100 (weeks) (years) Time Post Infection

What may HIV cure look like 1,000,000 Therapeutic vaccination 100,000 cART cLRAs 10,000 Plasma HIV RNA (copies/ml) 1000 100 (weeks) (years) Time Post Infection

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 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.

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 reservoir

Module collaborators We would like to thank all the module collaborators: Johns Hopkins University AVAC Collaboratory of AIDS Researchers for Eradication (CARE) and CARE Community Advisory Board (CAB)