MEASURING THE LATENT HIV RESERVOIR HIV Cure Research

MEASURING THE LATENT HIV RESERVOIR HIV Cure Research

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 Dub, 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 HIV persistence Cell Death Resting State Naive Establishment of immunologic memory Ag

Memory Naive Establishment of immunologic memory Ag

Memory Ag HIV infection of activated and resting CD4+ T cells Ag

HIV Naive Ag HIV Memory HIV Naive

Establishment of the latent reservoir in resting CD4+ T cells Ag Memory HIV 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 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 associated with smaller reservoirs 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 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: 1. PCR-based assays a. Quantitative PCR (qPCR) b. Reverse transcription PCR (rtPCR)

2. 3. 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

Measuring proviral DNA: PCR Quantitative PCR (qPCR) measures the amplification of DNA using fluorescence Fluorescence is proportional to the amount of PCR product probe (can bind to target nucleotides) fluoresce nt reporter quench er dye

Beacon. When reporter and quencher are close, quencher absorbs fluorescence Measuring proviral DNA: PCR Quantitative PCR (qPCR) measures the amplification of DNA using fluorescence Fluorescence is proportional to the amount of PCR product probe (can bind to target nucleotides)

fluoresce nt reporter quench er dye Beacon. When reporter and quencher are close, quencher absorbs fluorescence primer 1 Target PCR product primer 2

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 Measuring proviral DNA: PCR qPCR can be used to measure: 1. 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 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) 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 Chomont N 2014 at Towards and HIV Cure Symposium, IAS TILDA 1. Collect 10 to 20 mL of blood 2. Apply blood to Ficoll gradient centrifugation TILDA 1. Collect 10 to 20 mL of blood 2. Apply blood to Ficoll gradient centrifugation Blood sample Ficoll centrifuge

Plasma PBMCs Ficoll RBCs 3. Isolate CD4+ T cells from PBMC layer TILDA 4. Split isolated CD4+ T cells into two samples 5. Distribute both samples in limiting dilutions Plate 1 Plate 2 TILDA 6. 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 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 RNA Measuring the reservoir: VOA Viral Outgrowth Assay measures replicationcompetent HIV Provides a definitive minimal estimate of reservoir size Overview of process:

1. Resting CD4+ T cells are activated a. b. Resting cells do not produce virus without stimulation Activation reverses latency 2. Virus is expanded in cells from uninfected donors a. Added at two different time points 3. Assay is assessed by ELISA for p24 (viral protein) Ho, Cell 2013 Quantitative viral outgrowth assay 200 ml blood Purified resting CD4+ T cells

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 Cells are plated in dilution Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART

PURIFIED RESTING CD4+ T CELLS Ne co gati nt ve ro l 1. 6x 10 2 8x 10 3 4x 10 4

2x 10 5 10 6 5x 10 6 200 ml blood 1/1,000,000 Cells are plated in dilution Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART

PURIFIED RESTING CD4+ T CELLS Ne co gati nt ve ro l 1. 6x 10 2 8x 10 3 4x 10 4

2x 10 5 10 6 5x 10 6 200 ml blood 1/1,000,000 Cells are plated in dilution Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART Ne

co gati nt ve ro l 1. 6x 10 2 8x 10 3 4x 10 4 2x 10 5 10 6

5x 10 6 200 ml blood PURIFIED RESTING CD4+ T CELLS Resting CD4 T cells are activated using PHA. Since resting cells do not produce virus without stimulation, PHA is used to reverse latency. REACTIVATIO N WITH PHA Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART Ne

co gati nt ve ro l 1. 6x 10 2 8x 10 3 4x 10 4 2x 10 5 10 6

5x 10 6 200 ml blood PURIFIED RESTING CD4+ T CELLS ADD CD4+ FROM HIV NEG. DONOR REACTIVATIO N WITH PHA VIRUS AMPLIFICATION Latently infected cells can then then produce virus

which is expanded by add CD4+ T cells from HIV negative donors Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART Ne co gati nt ve ro l 1. 6x 10 2 8x 10 3

4x 10 4 2x 10 5 10 6 5x 10 6 200 ml blood PURIFIED RESTING CD4+ T CELLS ADD CD4+ FROM HIV NEG. DONOR

REACTIVATIO N WITH PHA ADD CD4+ FROM HIV NEG. DONOR After two weeks, add more HIV negative CD4+ Tcells VIRUS AMPLIFICATION Adapted from Finzi et al., Science, 1997 Quantitative viral outgrowth assay PATIENT ON ART PURIFIED RESTING

CD4+ T CELLS Ne co gati nt ve ro l 1. 6x 10 2 8x 10 3 4x 10 4 2x 10 5

10 6 5x 10 6 200 ml blood Can now grow out from single latently infected cell enough virus to detect with an ELISA HIV p24 Ag ADD CD4+ FROM HIV NEG. DONOR REACTIVATIO N WITH PHA

ADD CD4+ FROM HIV NEG. DONOR 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 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 - + 47% PHA+ allo PBMC

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

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

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

Resting CD4+ T cells PHA+ allo PBMC - 53% PHA+ allo PBMC - + 39% + 39% 61% PHA+ allo PBMC -

+ 47% 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

Scale=1/106 Ho et al, Cell, 2013 Katie Bruner, Nina Hosmane Model for time to rebound Hill et al, PNAS, 2014 What may HIV cure look like Plasma HIV RNA (copies/ml) 1,000,000 100,000 10,000 1000

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

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

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

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

cART cLRAs 10,000 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 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

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