a murine viral outgrowth assay to detect residual hiv-1 in patients with undetectable viral loads...
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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
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