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

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

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

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

Clinical samples 12 Acute/early participants 14 Chronic participants 1 1 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

Single-genome/proviral sequencing Sorted cells Lyse sorted cells 1 2 3 4 5 6 7 8 9 10 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

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

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

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

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

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

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

HIV persistence due to cellular proliferation START ART

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

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

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.

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