1. Conflict of Interest
No conflicts of interest to declare

2. Clinical Retrovirology Section, HIV DRP, NCI, NIH
Accumulation and Persistence of Deleted HIV Proviruses Following Prolonged ART
Elizabeth M. Anderson
Clinical Retrovirology Section, HIV DRP, NCI, NIH

3. HIV Persists Despite Suppressive ART
cART
1e5
HIV DNA (cps/1e6 CD4)
1e3
1e4
1e5
1e4
HIV RNA (cps/mL Plasma)
1e3 50
LOD
Mechanisms that maintain the reservoir from which viremia rebounds and is the target for elimination efforts
Time
Upon treatment initiation HIV decays: VL ~4-logs, HIV DNA declines ~20-fold
Low level plasma viremia persists in most patients, HIV DNA is stable
Mechanisms that maintain the reservoir are not well understood
Besson et al. & Mellors CID Palmer et al. & King PNAS 2008

4. Long Lived HIV Infected cells – A Major Barrier Towards Cure and Contributor to Pathogenesis
Potential mechanisms of reservoir maintenance
Homeostatic proliferation
Clonal expansion
Time on cART
Replication competent proviruses can clonally expand
Only a fraction of cells in the clone give rise to persistent viremia
Wagner et al. & Frenkel Science Maldarelli et al. & Hughes Science Josefsson et al. & Palmer 2013
Cohn et al. & Nussenzweig Cell Simonetti et al. & Maldarelli PNAS Symons et al. & Lewin IAS2017
Lee et al.& Palmer IAS Musick et al. & Kearney IAS 2017

5. Proviral landscape after prolonged cART
Long Lived HIV Infected cells – A Major Barrier Towards Cure and Contributor to Pathogenesis
Proviral landscape after prolonged cART
The majority of proviruses (>95%) are defective or deleted
Some may still produce protein
Could contribute to persistent immune activation
Bruner et al. & Siliciano Nat Med Imamichi et al. & Lane PNAS Pollack et al. & Ho Cell 2017
Imamichi IAS2017

6. How does the Proviral Landscape Change during cART?
Composition of HIV infected cells pretherapy
Proviral landscape after prolonged cART
Increased Ratio of LTR:gag
Years on cART
Approximately 2:1 Ratio of LTR:gag
What are the kinetics of this shift?

7. Quantifying HIV DNA to Resolve HIV Population Structure
ddPCR approaches for HIV DNA quantification
Multiplexed primer sets that span the HIV genome
Simultaneous quantification of LTR and internal HIV DNA
Accurate and able to resolve small (<2-fold) changes
Compared to qPCR

8. Participant Demographics
HIV-1 infected, therapy naïve individuals initiated cART (N=11)
Clinical trial starting regimen: AZT, 3TC, NVP, IDV
PID
Age at Entry
Baseline HIV RNA (Log10 cps/mL)
Baseline CD4 (cells/μl)
Longterm CD4 (cells/μl)
Days to Suppression
Years on ART 1 51
5.58 23
584 82
6.1 2 32
5.86
479
713
167
16.4 3 33
4.84
282
501
14.4 4 37
4.74
687
1206 62
9.8 5 25
4.31
258
488
6.0 6 34
4.53
373
478 26
15.5 7 29
4.43
440
484
111
13.0 8 50
4.88
572
385
14.1 9 39
5.10
283
278
280
16.1 10
4.78
379
895
140
15.9 11
5.38
 321
267
 62
15.7
Median
Integration Sites1
Relatively young with substaintial viral load RNA, One participant had AIDS and for this particular individual we have extensive Integration Site analysis data from which I will discuss more in the second part of my talk, On the other hand 3 participants were enrolled during relativly early infection within a year shown in blue, Participants responded to therapy showed typical decay patterns and were treated for a median of 14years.
The early infections were within a year- They were Feibig 5 when they were 1st sampled but were diagnosed within the year – time from diagnosis to sampling can be long due to enrollment procedures
PID 11 – had prostate cancer and was irradiated, during radiation CD4 dropped in half, the CD4% remained stable as previously reported.
1Maldarelli et al. & Hughes Science 2014

9. Sampling Strategy // // // I I I Pre-therapy
cART
1e5
1e5
RNA
1e4
1e4
HIV RNA cps/mL Plasma
HIV DNA
cps/1e6 CD4+
DNA
1e3
1e3 // 50 // I I // I
Pre-therapy
During 1st and 2nd phase viral decay
After Prolonged cART (Median: 14y, Range: 6y-16y)

10. HIV DNA and VL Decrease upon cART Initiation LTR:gag Ratio Increases after Prolonged cART

11. HIV LTR:gag DNA Ratio Increases after Prolonged cART in MOST but not ALL Patients
PID
001
002
003
004
005
006
007
008
009
010
011
6.1y
16.4y
14.4y
9.8y
6.0y
15.5y
13.0y
14.1y
16.1y
15.9y
15.7y
6.6
5.6
5.2
5.7
2.0
3.4
4.4
9.7
4.2
10.3
2.8
Pre-Therapy
Viral Decay
Prolonged
cART
Paired T-test
** p<0.01

12. Alteration of the Proviral Landscape Revealed by cART Why does the LTR:gag Ratio Increase?
Full Length
Deleted
Elimination of expressed proviruses (gag decreases)
Clonal expansion of deleted proviruses (LTRs Increase)

13. Highly Expanded Defective Clone Identified in PID001
ISA: highly expanded cell clone with and integrant in HORMAD2
Present at 20% after 6y on cART
Characterized as a solo LTR
Maldarelli et al. & Hughes Science 2014
Integration sites identified once
Forward Primer Overlapping HIV/Host Junction at the HORMAD2 Integration Site U3 R U5
HORMAD2
HIV LTR Specific Reverse and Probe
Simonetti Unpublished Data

14. Clonal Expansion of Deleted Proviruses can Contribute to a Shift in the Proviral Landscape
At 6 months HORMAD2 integrant is detectable and comprises around 8% of all integrants at 1 year on therapy the hormad 2 integrant doubles and by 2 years the integrant quadruples and now comprises of 30% of all integrants
Expansion of the integrant in HORMAD2 cannot be contributed to redistribution of CD4 alone, alternatively the cells that harbor the HORMAD2 integrant may be responding to an antigen. The fact that the integrant in HORMAD2 is then maintained for years on cART suggests that they are responding to a common antigen.

15. Summary
HIV LTR:internal DNA ratio increases in most patients after years on therapy indicating the accumulation of deleted proviruses
gag & tat/rev exon1&2
Specific deleted integrants (Solo LTR in HORMAD2) can undergo rapid clonal expansion and contribute to the shift in proviral landscape
Defective expanded clones can be maintained for years on therapy

16. Patient Participants Acknowledgments Clinical Retrovirology Section
Frank Maldarelli
Shawn Hill
Camille Lange
Monica Gouzoulis
Junko Hattori
Sarah Watters
Giorgio Bozzi
Francesco Simonetti
Jennifer Bell
Zehava Grossman
Translational Research Unit
Mary Kearney
Wei Shao
Valerie Boltz
Ann Wiegand
Jon Spindler
HMMC
Rob Gorelick
Mike Piatak
Tufts University
John Coffin
Catholic University of America
Venigalla Rao
John Choy
Ann Corsi
University of Pittsburgh
John Mellors
Patient Participants