I declare that I have no financial conflicts of interest Cytotoxic - - PowerPoint PPT Presentation

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

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?

Strategies to Improve Upon Antiretroviral Therapy

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?

Latency reversing drug HIV

Shock and Kill Approach to HIV Eradication

Cytotoxic T‐lymphocyte

Natural Reservoirs Contain Intact and Defective HIV

• What is the reservoir that matters?

Some Defective Proviruses Can Express Antigens

*** %CD107a

CAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEK

• -------D-------R-----------------------
• -*-----D-------R-----------G-L---------

........................................

• ------.--------R-----------------------

........................................

• -*-----D-------R-----------------------

EAAEWDRVHPVHAGPIAPGQ

• --------------T----
• ---*--L------------
• --------------T----

....................

• --D---L-------A----
• ------T-------V----

MGARASVLSGGELDRWEKIRLRPGGKKKYKL

• -----I----------------------Q-

I-----I------------------------

• -----I----------------------Q-

...............................

• -----I-R-EK—A----K--------H-M-
• OM5267

B27-Gag-IK9 IRLRPGGKK OM5011 B07-Gag-HA9 HPVHAGPIA OM5267 Cw08-Nef-AL9 AAVDLSHFL **** ** * 45E6 31G4 48C8 E44E11 4F12 19B3 Vector Peptide 100 10 1 100 10 1 45E6 31G4 48C8 E44E11 4F12 19B3 Vector Peptide %CD107a * 45E6 31G4 48C8 E44E11 4F12 19B3 Vector Peptide 100 10 1 %CD107a

HXB2 45E6 31G4 48C8 E44E11 4F12 19B3 Reference Ψ deletion Hypermutation Internal deletion Internal deletion Internal deletion Nonsense mutation HXB2 45E6 31G4 48C8 E44E11 4F12 19B3 HXB2 45E6 31G4 48C8 E44E11 4F12 19B3

Ya‐Chi Ho

• Data show CTLs responding to defective proviruses

Can CTLs Eliminate Intact / Defective HIV Reservoir

Directly from participant

1.6 87.3

No Peptide + HIV Peptide CD8 CD107a Copies HIV DNA/106 CD4+ Cells

HIV-Gag-HA9 CTL clone specificity

p < 0.0001

50 100 150 200 250

No Tx Gag-spec

• Bryo. +

Gag-spec CTL Bryo. p = 0.01

Subject OM5011 ddPCR

HIVE Results – ARV-Treated Subject #1

• Shock and kill – 50% reduction in HIV DNA
• 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)

0.01 0.1 1 10

Infectious Units Per Million

Subject OM5011 QVOA

N

• T

x B r y

• s

t a t i n B r y

• s

t a t i n + G a g

• s

p e c C T L

• Surprisingly, no decrease in infectious virus!

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

13.5 0.0 1.8 Uninfected Infected Infected + CTL HIV-Gag (Infected) CD4

OM5011 CTL Killing Assay

• Thus, failure to reduce intact-inducible virus not due to: i) immune escape

ii) lack of CTL cytotoxicity

+ +

• Infect activated

CD4+ T-cells with virus from single +QVOA well Co-culture for 16 hours and then measure % Infected (Gag+) by flow cytometry Co-culture infected targets with same CTL clone used in HIVE assay No CTL control

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 Allison Thomas John Huang Sara Karandish Dora Chan Adam Ward Collaborators Bruce Walker Darrell Irvine Douglas Nixon John Mellors Ya‐Chi Ho Robert Siliciano Clinical Samples Participants Colin Kovacs Erika Benko Mario Ostrowski Altor Bioscience Hing Wong Emily Jeng

PE-A

68.0

10 4 10 5 10 6

Alexa Fluor 700-A 102 103 104 105 PE-A

0.83

104 105 106 102 103 104 105

N

• T

x G a g

• s

p e c C T L V

• r

i n

• s

t a t V

• n

i n

• s

t a t + G a g

• s

p e c C T L

Vorinostat Cell-Associated HIV DNA

100 200 300

Copies HIV DNA/106 CD4+ Cells

CD8 CD107a

HIV-Gag-Spec CTL-HA9

No Peptide + Peptide

HIVE Results – ARV-Treated Subject #2 - Vorinostat

• No detectable decrease in cell-associated HIV DNA following

treatment with vorinostat + CTL

Cyt

Cytotoxic T‐Lymphocytes ‘CTL’ – Kill HIV Infected Cells

Sudha Kumari

CTL