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 & Funding Johns 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 AI and R21 AI National Institute of Mental Health grant P01 MH National Center for Research Resources Office of Research Infrastructure Programs grant P40 OD013117, K01 OD and T32 OD Johns Hopkins University Center for AIDS Research (CFAR) grant P30AI 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 L ) AbbVie (ritonavir) Janssen (darunavir) Most of this work has been published in Journal of Infectious Diseases on April 15 th, 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 PCRQVOA SensitivityHighestModerate Limiting factor to max # cells screened# obtained# feeders (10:1) Distinguish replication competency?NoYes 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 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 circulation Spearman R 2 = 0.87, P =

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? PatientsTotal testedTotal + by QVOATotal + by MVOA HIV+ Human on ART55 / 5 HIV+ Elite Suppressors65 / 66 / 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) PatientsTotal testedTotal + by QVOATotal + by MVOA HIV+ Human on ART55 / 5 HIV+ Elite Suppressors65 / 66 / 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 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 sensitivity 2.Serial dilutions in MVOA to determine IUPM 3.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

PCRQVOAMVOA SensitivityHighestModerateHigh(er?) Limiting factor to max # cells screened# obtained# feeders (10:1) # mice (1:50 mill) Distinguish replication competency?NoYesIt can Time to complete1 day2 weeksAve 3 weeks