1. The impact of treatment duration on defective and expanded identical HIV genomes in T cell subsets from peripheral blood and tissues
Eunok Lee

2. Antiretroviral therapy is not a cure
HIV persists during ART
Adapted from Kulpa and Chomont, Journal of Virus Eradication, 2015

3. Where does HIV hide?
HIV persists in several anatomic sites and different cellular subsets during ART.
Two arguments for how persistent HIV reservoirs are maintained during ART:
Ongoing low-level viral replication
Cellular proliferation
Searching for HIV reservoirs (anatomic and cellular levels) that contain replication competent HIV and understanding how the reservoirs are maintained during ART are important for the development of HIV eradication strategies.
Adapted from Barton et al., Trends in Microbiology, 2016

4. Questions to be addressed
Are Defective viruses enriched during effective therapy?
Differences between tissues and plasma
2) Which cellular and tissue compartments contribute to plasma viremia?
3) Are genetically identical viral populations enriched during effective therapy?
Differences between cells and tissues

5. Clinical samples 12 Acute/early participants 14 Chronic participants 11 3 18
-1.5
On-ART
Pre-ART
Years
28 Pre-ART plasma
Just before ART start &
1.5 years before ART start
19 on-ART plasma
mostly early ART
26 Peripheral blood
TN, TSCM, TCM, TTM, TEM
Lymph homing memory,
Gut homing memory
12 Lymph node tissue
TN, TCM, TTM, TEM
17 Gut tissue
TN, TCTM, TEM
CD4+ T cells
12 Acute/early participants
14 Chronic participants

6. Single-genome/proviral sequencing
Sorted cells
Lyse
sorted cells
endpoint dilution
Cell lysate/
plasma
Positive PCR
reactions
Purified DNA
PCR Amplify each p6-RT sequence
Purified RNA
cDNA
Pre-ART &
On-ART plasma
Align sequences
Gag-pol (p6-RT) sequences (1.2 kb)
Inter-patient analyses

7. Total of 4662 sequences derived from 26 participants
Total number of sequences analyzed
Total of 4662 sequences derived from 26 participants
Manuscript in preparation: PNAS

8. Number of sequences analyzed
participants treated during chronic infection
Total of 2808 sequences derived from 14 participants
who initiated ART during chronic infection
Manuscript in preparation: PNAS

9. No accumulation of defective HIV DNA sequences during ART
START
ART
Genetic diversity & defects
No evidence of ongoing low-level HIV replication in PB and tissues during 3-18 years of ART

10. Fewer defective HIV sequences were identified in pre- and on-ART plasma samples compared to anatomic regions
Pre-ART Plasma < PB
Pre-ART Plasma < LN
On-ART Plasma < PB
17-23%
On-ART Plasma < LN
6-10%
**** p<0.0001; *** p<0.001

11. Fewer defective HIV sequences were identified in pre- and on-ART plasma samples compared to CD4+ T cell subsets
6-10%
12-29%
Plasma PB LN
Gut
n≥4 are shown here
* p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001; *** p<0.001

12. HIV sequences from lymph node derived TEM are more often genetically identical to pre-ART and on-ART plasma  PB LN
Identical to Pre-ART PB LN
Identical to On-ART **
p=0.09
(n=9)
(n=7)
(n=7)
(n=6) ** **
p=0.09

13. HIV persistence due to cellular proliferation
START
ART

14. The number of identical HIV-DNA sequence expansions increases over time in peripheral blood but not in tissues

15. TEM from peripheral blood are the main contributor to the increase of identical HIV DNA sequences

16. Conclusions
The proportion of HIV proviruses that were defective did not increase during effective therapy.
Ongoing low-level viral replication is not a main cause for HIV persistence during therapy.
The defective HIV proviruses are established in cells prior to viral suppression.
Pre-ART and early on-ART plasma samples more often contained genetically intact HIV sequences than peripheral blood and tissues
HIV sequences from effector memory CD4+ T cells derived from lymph node were genetically similar to pre-ART and early on-ART plasma
Indicating that effector memory CD4+ T cells derived from lymph node mostly likely to contain replication-competent HIV in the region we sequenced.
Proliferation of HIV-infected T cells increased the proportion of identical HIV DNA sequences within peripheral blood over 3-18 years of therapy, and the EM cells were the main contributor.

17. We acknowledge with gratitude the participants of this study
Acknowledgements
Palmer lab WIMR: Centre for Virus Research
Associate Professor S. Palmer
S. von Stockenstrom
L. Odevall
K. Barton
B. Hiener
V. Morcilla
B. Horsburgh
A. Wincklemann
C. Wang
M-A. De Scheerder
K. Fisher
WIMR: Centre for Virus Research
Professor T. Cunningham
And Lv6 groups
University of Montreal
N.Chomont
R.Fromentin
Department of Epidemiology and Biostatics UCSF
P. Bacchetti
W. Hartogensis
Leidos Biomedical Research Inc. Frederick National Laboratory for Cancer Research
W. Shao
Immunology Laboratory, Vaccine Research Centre, NIH
E. Boritz
D. Douek
Department of Medicine UCSF
F. M. Hecht
S. G. Deeks
J. Milush
T. Liegler
M. Somsouk
P. Hunt
E. Sinclair
P. Lewis
H. Hatano
L. Epling
M. Kilian
T. Ho
A. Tan
J. Custer
L. Loeb
R. Hoh
L. Poole
We acknowledge with gratitude the participants of this study