Measuring the latent HIV Reservoir - PowerPoint Presentation

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

C ell D eath R esting S tate HIV persistence

Naive Ag † † † † † † Memory Establishment of immunologic memory

Naive Ag † † † † † † Ag † † † † † † Memory Establishment of immunologic memory

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

Establishment of the latent reservoir in resting CD4+ T cells Naive Memory Ag † † † † † † 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 20 th 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 20 th 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: 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 G rossly overestimate the size of the reservoir because they cannot distinguish defective vs. intact provirusMost 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 fluorescent reporter q uencher dye probe (can bind to target nucleotides) 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 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 fluorescence

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: 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 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 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, IAS

Collect 10 to 20 mL of blood Apply blood to Ficoll gradient centrifugation TILDA

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 entrifuge

TILDA Split isolated CD4+ T cells into two samples Distribute both samples in limiting dilutions 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 Plate 2 with PMA and ionomycin Nested PCR

TILDA Plate 1 Plate 2 + PMA and ionomycin Nested PCR

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 V iral O utgrowth A ssay measures replication-competent HIV Provides a definitive minimal estimate of reservoir sizeOverview of process:Resting CD4+ T cells are activatedResting cells do not produce virus without stimulationA ctivation reverses latencyVirus is expanded in cells from uninfected donors Added at two different time pointsAssay is assessed by ELISA for p24 (viral protein) Ho, Cell 2013

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 purified

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, 1997

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, 1997

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, 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 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, 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 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, 1997

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, 1997

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, 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 VOA Intact HIV DNA Scale=100/10 6 Ho et al, Cell, 2013

Size of the latent reservoir VOA Intact HIV DN A Scale=100/10 6 Ho et al, Cell, 2013

Size of the latent reservoir VOA Intact HIV DN A Scale=100/10 6 Ho et al, Cell, 2013

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

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

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 cells

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 cells

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 cells

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 6

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

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 )

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 )

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

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 cLRAs

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 cLRAs

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