A murine viral outgrowth assay to detect residual HIV-1 in patients with undetectable viral loads Kelly A. Metcalf Pate, DVM, PhD, DACLAM Joel N. Blankson,

A murine viral outgrowth assay to detect residual HIV-1 in

patients with undetectable viral loads

Kelly A. Metcalf Pate, DVM, PhD, DACLAM

Joel N. Blankson, MD, PhD

Johns Hopkins University School of Medicine, Baltimore, MD, USA

Acknowledgement of Co-Authors & FundingJohns Hopkins University School of Medicine, Baltimore, USA• Chris W. Pohlmeyer• Victoria E. Walker-Sperling • Jeremy B. Foote • Kevin M. Najarro • Catherine G. Cryer (now University of Pennsylvania School of

Veterinary Medicine)• Maria Salgado (now at Universitat Autònoma de Barcelona, Badalona,

Spain)• Elizabeth L. Engle • Erin N. Shirk • Suzanne E. Queen • Stanley Chioma • Meghan S. Vermillion • Claire E. Lyons (now at Cummings School of Veterinary Medicine at

Tufts University)• Brandon Bullock • Ming Li • Robert J. Adams • Lucio Gama • M. Christine Zink • Janice E. Clements • Joseph L. Mankowski

University of California San Francisco, San Francisco, USA• Hiroyu Hatano MD• Steve Deeks MD

Funding Sources• National Institute of Health (NIH)

• National Institute of Allergy and Infectious Diseases grants R56 AI080328 and R21 AI106491

• National Institute of Mental Health grant P01 MH070306 • National Center for Research Resources Office of

Research Infrastructure Programs grant P40 OD013117, K01 OD018244 and T32 OD011089

• Johns Hopkins University Center for AIDS Research (CFAR) grant P30AI094189

• Collaboratory of AIDS Researchers for Eradication (C.A.R.E.)

• Spanish Health Institute Sara Borrell grant • For generous donation of drugs for macaques

• Gilead (tenofovir)• Bristol-Myers Squibb (atazanavir)• Merck (integrase inhibitor L000870812)• AbbVie (ritonavir)• Janssen (darunavir)

Most of this work has been published in Journal of Infectious Diseases on April 15th, 2015

Quest for a Cure Needs Better Methods Viral Detection

Current methods of detection are not telling us when virus is eradicated

When is it safe to stop ART / other therapy?

• Harmful cytokine storm if viral resurgence

• Latent reservoir reseeded with every resurgence

Current Methods to Detect Residual Virus

• Sensitivity is an issue; inducible reservoir is larger than what is induced by QVOA (Ho et al Cell 2013)

• Need 10 times as many feeders as patient cells

• Need 4 times as many CD4 blasts as patients cells

• Have to add blasts twice

PCR QVOASensitivity Highest ModerateLimiting factor to max # cells screened # obtained # feeders (10:1)

Distinguish replication competency? No Yes

Courtesy of Bob Siliciano

QVOA

What the Eradication Effort Needs

1. An assay that is more sensitive than QVOA

2. Assay that can sample very large number of cells easily

3. An assay that selectively detects replication-competent virus

4. An assay that takes a reasonable amount of time to yield a result

How can we efficiently activate a large # of infected cells?

Hypothesis:Cellular activation caused by GVHD

secondary to adoptive transfer of infected cells from patients with undetectable

plasma viral loads into immunodeficient mice will result in amplification of HIV to a

detectable level

Mouse Viral Outgrowth Assay = MVOA

• NSG mice lack B, T and NK cells, engraft with human T cells but develop GVHD

• Inject 10-50 million PBMCs or CD4+ T cells from HIV-infected participants into each mouse intraperitoneally

• +/- activation with anti-CD3

• +/- depletion of CD8+T cells • Measure plasma viremia in mice through weekly

bleeds and in terminal bleed

Adoptive transfer by IP injection results in xenograft and activation of human cells in the NSG mouse

Also saw in GI, lymph nodes, liver and lungs

Activation noted as evidenced by CD25, HLA-DR & CD69

18 out of 18 mice humanized successfully

Correlation between # cells injected & % in circulationSpearman R2 = 0.87, P = 0.0005

Testing the MVOA

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART?2. HIV-1 infected elite suppressors?

Blankson et al JVI 2007

MVOA detects HIV-1 from patients on suppressive ART• Injected 25 to 55

million PBMCs from 5 patients on suppressive cART regimens (1 to 6 years) intraperitoneally (IP) into NSG mice

• On day 7 we gave anti-CD8 mAb IP

• Mice were bled weekly to evaluate viral load and CD4 T cell count

MVOA detects HIV-1 from Elite Suppressors• Injected 66 million PBMCs

from 1 ES OR 20 – 26 million purified CD4 T cells from ES IP into NSG mice

• On day 7 we gave anti-CD8 mAb IP for the mice engrafted with PBMCs

• For the mice engrafted with purified CD4s, we gave anti-CD8 as needed

• 2 mice were treated with anti-CD3 mAb (OKT3) IP

• Mice were bleed weekly to evaluate viral load and CD4 T cell count

Testing the MVOA

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

Blankson et al JVI 2007

Testing the MVOA: Comparison MVOA vs. QVOA

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

How does the sensitivity of the MVOA compare to the QVOA?

Patients Total tested Total + by QVOA Total + by MVOAHIV+ Human on ART 5 5 / 5 5 / 5HIV+ Elite Suppressors 6 5 / 6 6 / 6

MVOA detects HIV-1 from one more patient than QVOA

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

How does the sensitivity of the MVOA compare to the QVOA? High(er)

Patients Total tested Total + by QVOA Total + by MVOAHIV+ Human on ART 5 5 / 5 5 / 5HIV+ Elite Suppressors 6 5 / 6 6 / 6

Testing the MVOA: How Many Cells Can Screen?

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

How does the sensitivity of the MVOA compare to the QVOA? High(er)

How many cells can we efficiently screen with the MVOA?

MVOA can efficiently screen 350 million CD4+ T cells

• Viremic controller on cART• IUPM not determined by QVOA,

but assumed to be low (Chun T et al JID 2013)

• Obtained 1 billion cells from leukopak

• Isolated 350 million CD4+ T cells• Engrafted 7 NSG mice with 50

million CD4+ T cells each• Gave anti-CD8 as needed• Treated with anti-CD3 mAb

(OKT3) IP• Mice were bleed weekly to

evaluate viral load and CD4 T cell count

SFA1 SFA2 SFB1 SFB2 SFB3 SFB4 SFB5 1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

Patient 1242

VL

LOD

Mouse

Copi

es /

mL P

lasm

a HI

V-1

In collaboration with Hiroyu Hatano MD & Steve Deeks MD, UCSF

Testing the MVOA: How Many Cells Can Screen?

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

How does the sensitivity of the MVOA compare to the QVOA? High(er)

How many cells can we efficiently screen with the MVOA? >350 million CD4s

Testing the MVOA: How Many Cells Can Screen?

Will we amplify virus from patients with undetectable plasma viral loads?1. HIV-1 infected patients on suppressive cART? Yes!2. HIV-1 infected elite suppressors? Yes!

How does the sensitivity of the MVOA compare to the QVOA? High(er)

How many cells can we efficiently screen with the MVOA? >350 million CD4s

Are we amplifying patient virus with the MVOA?

Viral isolates from mice matched patient isolates• Sequenced virus

from plasma, spleen and/or peritoneal wash fluid from mice from 4 patients

• HIV-1 gag and nef sequence from isolates from mice shared homology with virus from each donor patient

The MVOA May Be What the Eradication Effort Needs

1. MVOA is at least as sensitive as the QVOA

2. MVOA can sample at least 350 million CD4+ T cells in one run

3. MVOA appears to be amplifying replication competent virus

4. MVOA takes a median of 26 days to yield results

Future directions:1. Continued comparisons with the

QVOA to further test sensitivity2. Serial dilutions in MVOA to

determine IUPM3. Additional comparisons of virus

harvested from the mouse to virus from the host

4. Further optimization of depletion and activation protocols to shorten detection time

5. Test ability to detect residual virus in tissue CD4+ T cells

Thank you for your attention

Questions?

Café Hon, Baltimore, USA

MVOA detects SIV from macaque on suppressive ART• Injected 40 million

PBMCs OR 6.8 million CD4+ T cells pigtailed macaque on suppressive cART regimens (78 days since last detectable viral load) IP into NSG mice

• Mice were bled weekly to evaluate viral load and CD4 T cell count

CD8+ T cells may control viremia in MVOA

PCR QVOA MVOASensitivity Highest Moderate High(er?)Limiting factor to max # cells screened # obtained # feeders(10:1) # mice (1:50 mill)

Distinguish replication competency? No Yes It canTime to complete 1 day 2 weeks Ave 3 weeks