Characterization of persistent HIV-1 in a broad spectrum of CD4 + T-cells isolated from peripheral blood, bone marrow, lymphoid tissue and gut associated lymphoid tissue from patients on long-term suppressive therapy Lina Josefsson Karolinska Institutet, Solna, Sweden “Towards an HIV Cure” Pre-Conference Symposium 20 & 21 July 2012

Background Persistent HIV can still be detected in plasma and cellular reservoirs even after several years of suppressive antiretroviral therapy Identifying the source and dynamics of persistent HIV in patients on suppressive therapy is an important step towards HIV eradication

Objectives of the study Use single-genome and single-proviral sequencing assays, to investigate the: – Source of persistent HIV-1 What cells are HIV reservoirs during suppressive therapy? – Dynamics of persistent HIV-1 Does low-level viral replication maintain HIV during suppressive therapy? 8 patients on suppressive antiretroviral therapy (4-12 years) – 5 initiating therapy during acute/early infection (< 6months) – 3 initiating therapy during chronic infection (> 1.5year) Peripheral blood (PB) Bone Marrow (BM) Gut Associated Lymphoid Tissue (GALT) Lymph Node Tissue (LN)

Layout of the study 19 oktober 2015Lina Josefsson4 Initiation of cART Plasma 1) Chronic: 1-3years Plasma 2) Chronic: 1-6 months Acute/early: 0-12 days Cells: Progenitor (CD34+ Lin-) Late progenitor (CD34-, Lin-) (CD34+, Lin-) Lymphoid cells (Lin+ CD4+) Naïve (CD45RO-CD27+) Central Memory (CD45RO+CD27+) Effector Memory (CD45RO+CD27-) Myeloid (CD3- CD4lo ) 5 patients treated during acute/early infection 3 patients treated during chronic infection 1.Investigate the source of persistent HIV-1 - Characterization of HIV-1 in infected cells after long-term suppressive cART 2.Investigate the dynamics of persistent HIV-1 - Compare pre-therapy HIV-1 populations to HIV-1 populations after long-term suppressive cART Treatment naive HIV-1 < 75 copies/ml 4 – 12 years LNPB GALT BM P6, protease, RT gag-pro-pol

Higher frequency of infection in cells isolated from patients treated during chronic infection compared to patients treated during acute/early infection Peripheral blood a p <0.05 * One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds. ** Numbers in parenthesis denotes the frequency of infection found in myeloid lysate with T-cell receptors present. *** For calculation of geometric means, estimated infection rates of zero were replaced with 1/(number of cells tested) 0.007% 0.06% X9 0.01% 0.03% X3 0.01% 0.13% X13 a % 0.01% X24 a

Gut Associated Lymphoid Tissue Higher frequency of infection in cells isolated from patients treated during chronic infection compared to patients treated during acute/early infection * One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds. ** Numbers in parenthesis denotes the frequency of infection found in myeloid lysate with T-cell receptors present. *** For calculation of geometric means, estimated infection rates of zero were replaced with 1/(number of cells tested) 0.009% 0.03% 0.02% 0.15% 0.07% 0.08% X4X6 a X1.2 a p <0.05

No infected progenitor cells isolated from bone marrow * One cannot rule out the possibility of low but nonzero infection rates in cases where estimates were zero, as shown by the upper confidence bounds. Bone marrow a p < 0.05 Josefsson et al, JID 2012 X13 a

Phylogenetically indistinguishable HIV-1 sequences from plasma collected before initiation of cART and cells isolated after several years of suppressive therapy Patient 4 Treated during early infection > 6 years on therapy Peripheral blood – Central memory Peripheral blood – Effector memory On-therapy Peripheral blood – All memory Peripheral blood – Naive Pre-therapy Plasma GUT – Effector memory Lymph node – Effector memory Lymph node– Transitional memory Bone marrow – Lin+CD

Phylogenetically indistinguishable HIV-1 sequences from plasma collected before initiation of cART and cells isolated after several years of suppressive therapy Patient 8 Treated during chronic infection >8 years of therapy Peripheral blood – Central memory Peripheral blood – Effector memory On-therapy Peripheral blood- All memory Peripheral blood – Naive Pre-therapy Plasma GUT – Effector memory GUT – Central memory GUT– Naive Lymph node– Effector memory Lymph node – Central memory Lymph node– Transitional memory Lymph node– Naive Bone marrow – Lin+CD4+ Bone marrow – Lin-CD

No clear detection of viral evolution between sequences isolated from pre-therapy plasma and sequences isolated from peripheral blood or GALT collected after 4-12 years of suppressive cART 3 different approaches to investigate the amount of viral evolution between sequences from plasma before the initiation of therapy and sequences from cells after 4-12 years of suppressive cART Genetic distance Correlation of genetic divergence and time Evolutionary rate analysis Genetic distance between: 1. Sequences from pre-therapy and on-therapy 2.Sequences from pre-therapy 3.Sequneces from on-therapy Correlation of genetic divergence and time between: 1. Pre-therapy and on-therapy 2.Pre-thearpy (1) and pre-therapy(2) for patients treated during chronic infection Nucleotide substitutions per site per year between: 1. Pre-therapy and on-therapy 2. Pre-therapy (1) and pre-therapy(2) for patients treated during chronic infection Comparison of genetic distance between sequences from the same time point differ no more than the comparision of sequences between PT and OT indicating no evolutionary change during suppressive therapy No correlation between genetic divergence and time between pre-therapy and on-therapy sequences indicating no evolutionary change during suppressive thereapy. Correletion between pre-therapy (1) and pre-thearpy (2). Nucleotide substitutions per site per year: 1. Patients treated during early infection: mean = 10e-5 2. Patients treated during chronic infection: mean = 10e-4 3. Pre-therapy sequences from patients treated during chronic infection mean = 10e-3 Genetic distance Genetic distance between: 1. Sequences from pre-therapy and on-therapy 2.Sequences from pre-therapy 3.Sequneces from on-therapy Comparison of genetic distance between sequences from the same time point differ no more than the comparison of sequences between PT and OT indicating no evolutionary change during suppressive therapy Correlation of genetic divergence and time Correlation of genetic divergence and time between: 1. Pre-therapy and on-therapy 2.Pre-thearpy (1) and pre-therapy(2) for patients treated during chronic infection No correlation between genetic divergence and time between pre-therapy and on- therapy sequences indicating no evolutionary change during suppressive therapy. Correlation between pre-therapy (1) and pre-therapy (2). R 2 = – Evolutionary rate analysis Nucleotide substitutions per site per year between: 1. Pre-therapy and on-therapy 2. Pre-therapy (1) and pre-therapy (2) for patients treated during chronic infection Nucleotide substitutions per site per year: 1. Patients treated during early infection: mean = 10e-5 2. Patients treated during chronic infection: mean = 10e-4 3. Pre-therapy sequences from patients treated during chronic infection mean = 10e-3 Nucleotide substitutions per site/year: Early infection: 10e-5 Chronic infection: 10e-4 Pre-therapy: 10e-3

Conclusions T-cells were found to be infected in all patients after long-term suppressive therapy. No progenitor cells were found to be infected and myeloid cell samples contained T-cell receptors Early initiation of effective therapy results in lower reservoir size No clear evidence for evolution could be detected in patients treated during early and chronic infection The lack of clear evidence for HIV-1 genetic evolution in the HIV-1 infected CD4+ T-cell populations after years of therapy argues against virus replication as a major cause of persistence in these cell populations. – The role of replication in other tissues and cell types however remains to be defined.

Acknowledgements We acknowledge with gratitude the participation of all the patients in this study University of California, San Francisco F. Hecht E. Sinclair P. Bacchetti P. Lewis P. Hunt M.Somsouk H. Hantano S. Deeks L. Epling M. Kilian T. Ho A. Tan J. Custer L. Loeb R. Hoh L. Poole Karolinska Institutet The Swedish Institute for Communicable Disease Control S. Palmer S. Eriksson V. Dahl M. Mild J. Albert Rega Institute, Belgium N. Faria P. Lemey SAIC/NCI W. Shao VRC/NIAID D. Douek W. Wylie

Patient information Patient Viral Load Pre Therapy (RNA copies/ml) Viral Load On Therapy* (RNA copies/ml) CD4+ T-cell Count Pre Therapy (cells/ul) CD4+ T-cell Count On Therapy (cells/ul) Lenght of infection (years) Time on therapy (years) Treated during acute/early infection < < < < < Treated during chronic infection < < < * Viral RNA levels at the time of the study

Results – genetic distance Patient Genetic distance (%) (Pre-therapy sequences and on- therapy sequences) Genetic distance (%) (Pre-therapy sequences) Genetic distance (%) (On-therapy sequneces) Initiated therapy during acute/early infection Initiated therapy during chronic infection 6*0.87N/A *5.51N/A *1.43N/A * Pre-therapy samples collected 1-3 years apart

Results – regression analysis Patient Correlation of genetic divergence and time R2R2 Initiated therapy during acute/early infection 12.78E E E E E-02 Initiated therapy during chronic infection 6* E-03 7* * * Pre-therapy samples collected 1-3 years apart

Results – evolutionary rate analysis Treated during early infection Treated during chronic infection

0.01

18 Sort by cell type using FACS Single-proviral Sequencing PCR Amplify each HIV genome from the lysate and sequence P6, protease, RT gag-pro-pol Calculate genetic relationship to plasma sequences from sample taken before and after long-term therapy Align sequences Patient Cells from peripheral blood, bone marrow, lymphoid tissue and GUT Lyse sorted cells DNA endpoint dilution Cell lysate Positive PCR reactions Josefsson et. al. JID 2012

19 oktober 2015Namn Efternamn19 Multiple Alignment File cDNA endpoint dilution Agarose gel P6, protease, RT gag-pro-pol Sequence individual genomes Palmer et. al. J Clin Micro 2005 Kearney et. al. J Virol 2008 Single-Genome Sequencing of Plasma Samples