2014 “ Towards an HIV Cure ” symposium Melbourne

2014 “Towards an HIV Cure” symposiumMelbourne

A novel assay that precisely measures the size of the latent HIV reservoir reveals that ART-naive individuals harbour a large pool

of latently infected CD4+ T cells

Nicolas Chomont, PhD

Measuring the size of the reservoir

There is a need to develop sensitive, reproducible and clinical trial scalable methods to measure the size of the latent HIV reservoir

There is currently no gold standard method to measure the size of the latent HIV reservoir (frequency of latently infected cells)

•The quantitative viral outgrowth assay measures replication-competent HIV but may largely underestimate the size of the reservoir (Ho, Cell 2013)

•PCR based assays are reproducible, relatively easy to perform but may overestimate the size of the reservoir (Eriksson, Plos Pathogens 2013)

Defective viral genomes

Ho et al. Cell 2013

Principle of TILDA

TILDA: “Tat/Rev Induced Limiting Dilution Assay”

Nested RT-PCRfor msHIV RNA(24+40 cycles)

Maximum likelihood

methodFrequency of

cells with inducible msHIV

RNA

10-20 mL whole blood

PBMCs

Ficoll gradient centrifugation

CD4+

T cells

Negative selection

12h PMA+ionomycin

Distribute in limiting dilutions

Frequency of cells with msHIV

RNA baseline

TILDA in CD4 T cells from ART subjects

The majority of cells with inducible virus are latently infected in ART subjects

TILDA and other assays

TILDA gives a reservoir size in between Q-VOA and DNA

Adapted from Eriksson et al. 2013

TILDA and other assays

Total DNA in PBMCs Total DNA in resting CD4 Integrated DNA in resting CD4Integrated DNA in PBMCs

Total DNA in rectal CD4 Q-VOA SCA

TILDA correlates with several assays measuring HIV persistence

ART in Acute and Chronic infection

TILDA distinguishes between subjects who have started ART during acute and chronic infection

TILDA in viremic subjects (no ART)

The majority of cells with inducible virus are latently infected in ART naive subjects

TILDA in viremic subjects (no ART)

75% of the cells with inducible HIV are latently infected in untreated HIV infected subjects

ART VIR

Pre-integration latency?

Pre-integration latency

Post-integration latency

Entry Uncoating

Reverse transcription

Integration

Viral transcription

Viral production

Pre-integration latency?

Pre-integration latency

Post-integration latency

PMA+ionomycin

PMA+ionomycin

TILDA

+

+

RALTEGRAVIR(4h) PMA+ionomycin

+ RALTEGRAVIR

RALTEGRAVIR(4h) PMA+ionomycin

+ RALTEGRAVIR

+

-

TILDA with raltegravir

VIR1 VIR9 VIR11

Post-integration latency is already established in untreated HIV-infected subjects

Conclusions

TILDA is:• sensitive (1.4 cells/million)• reproducible (coefficient of variation <0.2)• fast (<2 days)• relatively inexpensive ($300)• easily transferable (basic culture set up + real time PCR) • Clinical trial transferable (requires only 10mL of blood)

The median frequency of “reservoir” cells measured by TILDA is 24 cells/million, which is 48 times more than Q-VOA and 6 to 27 times less than PCR-based assays

In untreated disease, the frequency of latently infected cells largely exceeds the frequency of productively infected cells suggesting that the majority of infected cells are transcriptionally silent even in the absence of ART

This provides a rationale for the use of shock and kill strategies at the time of ART initiation

Shock and kill at ART initiation

shock

HIV-specific CD8 T cells

Latent reservoir

HIV viral load

ART

ART

Acknowledgements

VGTI Florida

Francesco Procopio

Remi Fromentin

Deanna Kulpa

Amanda McNulty

Anne-Gaelle Blackwell

Lydie Trautmann

Rafick-Pierre Sekaly

Merck and Co

Daria Hazuda

Mike Miller

Richard Barnard

UCSF

Steven Deeks

Rick Hecht

Westmead Institute

Sarah Palmer

Karolinska Institutet

Susanne Eriksson

University of Pennsylvania

Una O’Doherty

Johns Hopkins

Robert Siliciano

Janet Siliciano

UCSD

Doug Richman

Matt Strain

The study participants!

U19AI096109 R21AI113096

ARCHE 108687-54