I declare that I have no financial conflicts of interest
Cytotoxic T-Lymphocytes Eliminate Defective HIV Proviruses Without Impacting Infectious Reservoirs R. Brad Jones Assistant Professor The George Washington University
Strategies to Improve Upon Antiretroviral Therapy 1) Sterilizing cure – eradicate all viral reservoirs from the body – stop antiretroviral therapy 2) Functional cure – enable long-term immune control of virus – stop antiretroviral therapy
Strategies to Improve Upon Antiretroviral Therapy 1) Sterilizing cure – eradicate all viral reservoirs from the body – stop antiretroviral therapy 2) Functional cure – enable long-term immune control of virus – stop antiretroviral therapy 3) Reduce HIV proviral (DNA) burden – continue with antiretroviral therapy but less inflammation? Improved health/quality of life?
HIVE Results – ARV-Treated Subject #2 - Vorinostat • Trend towards decrease in vorinostat only and vorinostat + CTL conditions – but no additive ‘kick and kill’ effect
Shock and Kill Paradigm • Landmark study - vorinostat alone did not drive reductions in infectious viral reservoirs from ex vivo CD4+ T-cells (natural reservoirs) Shan et al, 2012 • Can combinations of CTLs with LRAs drive reductions in natural reservoirs?
Shock and Kill Approach to HIV Eradication Latency reversing Cytotoxic drug T‐lymphocyte HIV
Natural Reservoirs Contain Intact and Defective HIV • What is the reservoir that matters?
Some Defective Proviruses Can Express Antigens • Data show CTLs responding to defective proviruses OM5267 OM5011 OM5267 B27-Gag-IK9 B07-Gag-HA9 Cw08-Nef-AL9 IRLRPGGKK HPVHAGPIA AAVDLSHFL Reference HXB2 HXB2 HXB2 MGARASVLSGGELDRWEKIRLRPGGKKKYKL EAAEWDRVHPVHAGPIAPGQ CAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEK Ψ deletion 45E6 45E6 45E6 ------I----------------------Q- ---------------T---- --------D-------R----------------------- Hypermutation 31G4 31G4 31G4 I -----I------------------------ ---- * --L------------ -- * -----D-------R-----------G-L--------- Internal deletion 48C8 48C8 48C8 ---------------T---- ........................................ ------I----------------------Q- Internal deletion E44E11 E44E11 E44E11 .................... -------.--------R----------------------- ............................... Internal deletion 4F12 4F12 4F12 ---D---L-------A---- ........................................ ------I-R-EK—A----K--------H-M- Nonsense mutation 19B3 19B3 19B3 -- * -----D-------R----------------------- ------------------------------- -------T-------V---- **** 100 100 100 ** * *** * %CD107a %CD107a %CD107a 10 10 10 1 1 1 E44E11 19B3 E44E11 19B3 E44E11 19B3 45E6 31G4 48C8 4F12 Vector Peptide 45E6 31G4 48C8 4F12 Vector Peptide 45E6 31G4 48C8 4F12 Vector Peptide Ya‐Chi Ho
Can CTLs Eliminate Intact / Defective HIV Reservoir Directly from participant
HIVE Results – ARV-Treated Subject #1 • Shock and kill – 50% reduction in HIV DNA HIV-Gag-HA9 CTL clone specificity Subject OM5011 ddPCR p < 0.0001 No Peptide + HIV Peptide Copies HIV DNA/10 6 CD4 + Cells 87.3 1.6 250 p = 0.01 200 150 CD107a 100 50 CD8 0 No Tx Gag-spec Bryo. Bryo. + Gag-spec CTL • Need elimination of defective proviruses to account for these decreases
HIVE Results – ARV-Treated Subject #1 • How much infectious virus is left in these cells (quantitative viral outgrowth assays) • Surprisingly, no decrease in infectious virus! Subject OM5011 QVOA 10 Infectious Units Per Million 1 0.1 0.01 n + L x i T t n T a C i t t o s a c N o t e s y p o r B y s - r B g a G
CTLs Eliminated Defective but not Intact Virus
Maximal Latency Reversal ‘Strongest Shock’ • Again, decrease in HIV DNA but no change in infectious virus
No CTL Escape Mutations in Infectious Virus Uninfected Infect activated - - + CD4+ T-cells with OM5011 CTL virus from single Killing Assay - - + Co-culture infected targets with same +QVOA well CTL clone used in HIVE assay 0.0 Infected Infected + CTL Co-culture for 16 hours and then measure % Infected (Gag+) by flow cytometry CD4 13.5 1.8 No CTL control HIV-Gag (Infected) • Thus, failure to reduce intact-inducible virus not due to: i) immune escape ii) lack of CTL cytotoxicity
Similar Results with Other CTLs and “Shocks” Cell-associated HIV DNA Quantitative Viral Outgrowth Assays
Decrease in HIV DNA But No Delay in Viral Rebound
Conclusions • “Shock and Kill” reduced defective HIV DNA but not infectious virus • Precision of assay to measure infectious virus is limited, but need much greater decreases to delay viral rebound • Defective HIV DNA can stimulate the immune system! Is this contributing to ongoing inflammation • Potential benefit of reducing HIV DNA, even if ARV therapy must be continued
Strategies to Improve Upon HAART 1) Sterilizing cure – eradicate all viral reservoirs from the body – stop antiretroviral therapy 2) Functional cure – enable long-term immune control of virus – stop antiretroviral therapy 3) Reduce HIV proviral (DNA) burden – continue with antiretroviral therapy but less inflammation? Improved health/quality of life?
Acknowledgments Lab Members Collaborators Clinical Samples Allison Thomas Bruce Walker Participants John Huang Darrell Irvine Colin Kovacs Sara Karandish Douglas Nixon Erika Benko Dora Chan John Mellors Mario Ostrowski Adam Ward Ya‐Chi Ho Altor Bioscience Robert Siliciano Hing Wong Emily Jeng
HIVE Results – ARV-Treated Subject #2 - Vorinostat HIV-Gag-Spec CTL-HA9 + Peptide 68.0 Vorinostat 105 Copies HIV DNA/10 6 CD4 + Cells Cell-Associated HIV DNA 300 104 PE-A 103 200 102 10 4 10 5 10 6 0 100 No Peptide 0.83 Alexa Fluor 700-A 105 0 104 PE-A x + L L T T t t T a a o C C t t N s s CD107a 103 c o c o e n e n p i p i r n s s o o - V - g V g a a 102 G G 104 105 106 0 CD8 • No detectable decrease in cell-associated HIV DNA following treatment with vorinostat + CTL
� � Cytotoxic T‐Lymphocytes ‘CTL’ – Kill HIV Infected Cells Cyt CTL Sudha Kumari
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