MEASURING THE LATENT HIV RESERVOIR HIV Cure Research

MEASURING THE LATENT HIV RESERVOIR HIV Cure Research Training Curriculum Scientific Leads: Janet Siliciano, Ph. D and Robert Siliciano, MDPh. D, 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

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 CD 4 T cells Ag HIV Naive † Ag HIV Memory † HIV

Naive Establishment of the latent reservoir in resting CD 4+ 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 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: 1. PCR-based assays a. b. Quantitative PCR (q. PCR) Reverse transcription PCR (rt. PCR) TILDA assay 3. Quantitative Viral Outgrowth Assay (QVOA) Gold Standard 2.

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 (q. PCR) measures the amplification of DNA using fluorescence • Fluorescence is proportional to the amount of PCR product probe (can bind to target nucleotides) fluorescent reporter quenche r dye Beacon. When reporter and quencher are close, quencher absorbs fluorescence

Measuring proviral DNA: PCR • Quantitative PCR (q. PCR) measures the amplification of DNA using fluorescence • Fluorescence is proportional to the amount of PCR product probe (can bind to target nucleotides) fluorescent reporter quenche r dye Beacon. When reporter and quencher are close, quencher absorbs fluorescence primer 1 Target PCR product primer 2

Measuring proviral DNA: PCR • Quantitative PCR (q. PCR) 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 q. PCR 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 only works on DNA • Reverse transcription PCR (rt. PCR) used to measure free virus and virus gene expression. • • RNA (from virus) is reverse transcribed into c. DNA The standard viral load assay is an rt. PCR 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 Vo. A 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 m. L of blood 2. Apply blood to Ficoll gradient centrifugation

TILDA 1. Collect 10 to 20 m. L of blood 2. Apply blood to Ficoll gradient centrifugation Blood sample Ficoll centrifuge Plasma PBMCs Ficoll RBCs 3. Isolate CD 4+ T cells from PBMC layer

TILDA 4. Split isolated CD 4+ 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 CD 4+ 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 ms. HIV RNA (baseline) Results from Plate 2 (stimulated with PMA + ionomycin) Frequency of cells with inducible ms. HIV RNA

Measuring the reservoir: VOA • Viral Outgrowth Assay measures replicationcompetent HIV • Provides a definitive minimal estimate of reservoir size • Overview of process: 1. Resting CD 4+ T cells are activated 2. Virus is expanded in cells from uninfected donors 3. Assay is assessed by ELISA for p 24 (viral protein) a. b. Resting cells do not produce virus without stimulation Activation reverses latency a. Added at two different time points Ho, Cell 2013

Quantitative viral outgrowth assay 200 ml blood Purified resting CD 4+ T cells Blood is drawn and resting CD 4+ T cells are purified Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay PATIENT ON ART 200 ml blood PURIFIED RESTING CD 4+ T CELLS Cells are plated in dilution Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay PURIFIED RESTING CD 4+ T CELLS 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART 1/1, 000 Cells are plated in dilution Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay PURIFIED RESTING CD 4+ T CELLS 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART 1/1, 000 Cells are plated in dilution Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART PURIFIED RESTING CD 4+ T CELLS Resting CD 4 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 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART PURIFIED RESTING CD 4+ T CELLS ADD CD 4+ FROM HIV NEG. DONOR REACTIVATION WITH PHA VIRUS AMPLIFICATION Latently infected cells can then produce virus which is expanded by add CD 4+ T cells from HIV negative donors Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART PURIFIED RESTING CD 4+ T CELLS ADD CD 4+ FROM HIV NEG. DONOR REACTIVATION WITH PHA ADD CD 4+ FROM HIV NEG. DONOR After two weeks, add more HIV negative CD 4+ Tcells VIRUS AMPLIFICATION Adapted from Finzi et al. , Science, 1997

Quantitative viral outgrowth assay PURIFIED RESTING CD 4+ T CELLS 10 2 6 x 1. 10 3 8 x 10 4 4 x 10 5 2 x 10 6 5 x 10 6 200 ml blood Ne co gat nt ive ro l PATIENT ON ART Can now grow out from single latently infected cell enough virus to detect with an ELISA HIV p 24 ADD CD 4+ FROM HIV NEG. DONOR REACTIVATION WITH PHA Ag ADD CD 4+ FROM HIV NEG. DONOR VIRUS AMPLIFICATION Adapted from Finzi et al. , Science, 1997

Technical challenges in measuring the reservoir • Latently infected resting CD 4+ 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 CD 4+ T cells Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD 4+ T cells - + 47% PHA+ allo PBMC 53% Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD 4+ 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 CD 4+ T cells PHA+ allo PBMC - 53% PHA+ allo PBMC - + 39% 61% PHA+ allo PBMC - + 47% 61% Ho et al, Cell, 2013 Nina Hosmane

Can intact non-induced proviruses be induced? Resting CD 4+ T cells PHA+ allo PBMC - 53% PHA+ allo PBMC - + 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, 000 100, 000 1000 100 (weeks) (years) Time Post Infection

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

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

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

What may HIV cure look like Plasma HIV RNA (copies/ml) 1, 000 100, 000 Therapeutic vaccination c. ART 10, 000 c. LRAs 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