366 Clues to Eradicate HIV
1
A Tale of Two Stem Cell Transplantations in HIV+ Patients: Clues to Eradicate HIV Background: The EpiStem Consortium Contact at: Maria Salgado [email protected] http://www.epistem-project.org/ Poster Number: 366 1 AIDS Research Institute irsiCaixa, Badalona, Spain; 2 Hospital Gral. Univ. Gregorio Marañon, Madrid, Spain; 3 University Medical Center Utrecht, Utrecht, The Netherlands; 4 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5 Cellex, Dresden, Germany; 6 ICREA, Barcelona, Spain. Maria Salgado 1 , M Kwon 2 , M Nijhuis 3 , J van Lunzen 4 , Julià Blanco 1 , J Schulze zur Wiesch 4 , G Hutter 5 , AM Wensing 3 , JL Diez 2 , Javier Martinez-Picado 1,6 for the EpiStem Consortium Results Conclusions Findings Methods Herein we have compared two EpiStem pa- tients with >20 months of follow up after transplantation. We observed how varia- tions in the transplantation proccess results in important differences in reservoir size. Patient 1 (Pt1) and Patient 3 (Pt3) were transplanted in Hospital Gregorio Marañón, Madrid, Spain. Samples were shipped to irsiCaixa (Barcelona), UMC Utrecht and UMC Hamburg-Eppendorf and analyzed for reservoir and immunological studies. - qVOA: 90 and120 Million bulk CD4+ T cells were used for the qVOA of patient 1 and 3 respectively. - HIV DNA was quantified using ddPCR in: -CD4+ T cells from peripheral blood leukapheresis -CD3+ T cells from Bone Marrow -CD4+ Cells from Ileum biopsy sorted using FACSaria II (Figure 4) - Semiquantitative PCR was performed using Gag prim- ers with limited dilutions of CD4 T cells DNA. - Susceptibility assay: CD4+ T cells from the patient were infected in vitro with R5 and X4 tropic viruses. - Ultrasensitive viremia: 9ml of plasma and 2.5ml of CSF were ultracentrifuged and HIV-RNA quantified using Abbott VL platform. - Chimerism was performed by short tandem repeat PCR (STR-PCR). - Antibody titers were determined by ELISA - Activation markers (CD38 and HLA-DR) were deter- mined in CD4+ and CD8+ T-cells. Acknowledgements QVOA Pre-SCT Post-SCT 10 -3 10 -2 10 -1 10 0 10 1 IUPM (CD4 T cells) Ultrasensitive Viremia (Blood) Pre-SCT Post-SCT 10 -1 10 0 10 1 10 2 10 3 Total HIV-1 RNA (copies/ml) Reservoir in GALT Pre-SCT Post-SCT 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 Total HIV-1 DNA (cells/million CD4+) Reservoir in Blood Pre-SCT Post-SCT 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 Total HIV-1 DNA (cells/million CD4+) EpiStem: Observational study Immunology Clinical Hematology BM and CB Donor Testing Evaluation and Counceling Virology To date, Berlin patient's case provides the only evidence of long-term HIV-1 control after allogeneic stem cell transplantation (SCT), potentially due to the CCR5Δ32/Δ32 donor genotype. The case of the two Boston patients, without resulting in HIV cure, showed that the SCT procedure itself involves a tremendous reduction of the viral reservoir. The reason why this happened is unraveled yet. EpiStem is a collaborative project to guide and investigate the potential for HIV cure in HIV-infected patients requiring allogeneic stem cell transplantation for hematological disorders. The EPISTEM consortium is composed of an expert panel of hematologists with experience in allogeneic SCT procedures along with infectious disease specialists, virologist and immunologists with expertise in the field of HIV-1, reservoirs and cure (Figure 1) . Principal achievements of the EpiStem Consortium: 1. EpiStem is the largest international cohort of allogeneic SCT recipients with HIV-1 infection currently including 14 transplanted patients (Figure 2) , 4 of them with CCR5Δ32/Δ32 donor . 2. Longitudinal samples from peripher- al blood (phlebotomy and leukapheresis), gut biopsy, cerebrospinal fluid (CSF), lymph node and bone marrow have been collected and analyzed. 3. In parallel, 30000 cord blood units have been tested for CCR5Δ32/Δ32 muta- tion in Europe. 200 mutated units have been identified and are available for new transplantations 4. Preliminary analysis of the cohort sug- gests that allogeneic stem cell transplan- tation resulted in a decay of the latent res- ervoir in all analyzed cases. (Figure 3) . Figure 1. EpiStem Consortium Figure 2. EpiStem Patients Figure 3. HIV reservoir after SCT NK-NHL HIV-1+ X4 tropic Patient 3 HLA-matched sibling (10/10) 2013 - Full Chimera (30 days) - GvH disease +20 month post SCT Reduced intensity conditioning Peripheral Blood Progenitor Cells SCT CCR5 wt - no cART interruption Patient 3 - no cART interruption Myeloablative Haplo-cord SCT CCR5 wt Burkitt NHL HIV-1+ R5 tropic Patient 1 Cord Blood (5/6) CD34+ HLA-mismatched donor 2012 - Chimera 0.2% BM/ 0.1% PB - No GvH disease +29 month post SCT Patient 1 - qVOA (IUPM) - Total DNA (cop/10 6 CD4) - Susceptibility (R5 and X4 viruses) - Immune supression - Activation Markers (HLA-DR+ CD38+ cells) - SCA (HIV-RNA cop/ml) - Ab titers - Ileum CD4+ cells - BM CD3+ cells - CSF CD4+ T cells Plasma Tissue Allogeneic SCT with Peripheral Blood Progenitor Cells of an HLA-matched sibling donor, with a faster achievement of full donor chimera and a chronic GvH disease resulted in a more drastic reduction of the latent reservoir down to undetectable levels. We hypothesize that the “graft versus HIV-1 reservoir effect” contributes to facilitate the clearance of the viral reservoir. We would like to thank all the collaborators, doctors, clinical staff and researchers from the EpiStem Consortium hospitals and research centers that have participated somehow to the sample collection and processing (listed at www.epistem-project.org). We also thank to the founders: amfAR (Arche program) and The Spanish ministry of Economy CD45 FITC CD3 APC CD8 PercP Figure 4. Ileum sorting strategy 0.034 25 Susceptible 69% of CD8+ inhibition CD4: 11.30% CD8: 19% 5 Low levels N/A N/A N/A Undetectable Undetectable Susceptible Non Effective CD8+ T response CD4: 1.41% CD8: 7.3% Undetectable Very Low levels DNA Undetectable DNA Undetectable RNA Undetectable P t 1 P t 2 P t 3 P t 4 P t 5 P t 6 0 P t 7 P t 8 P t 1 0 P t 1 1 Blood Apheresis Ileum biopsies CSF Bone Marrow P t 1 2 Months post-transplantaon 5 10 15 25 20 30 35 P t 1 7 60 P t 9 P t 2 0 CCR5 WT/WT CCR5 Δ32/Δ32 Adult Donor 9* 2 11 Umbilical cord 1 2 3 10 4 *CCR5 Δ32/WT CD4 cells
Transcript of 366 Clues to Eradicate HIV
A Tale of Two Stem Cell Transplantations in HIV+ Patients:Clues to Eradicate HIV
Background: The EpiStem Consortium
Contact at: Maria Salgado
[email protected]://www.epistem-project.org/
Poster Number: 366
1AIDS Research Institute irsiCaixa, Badalona, Spain; 2Hospital Gral. Univ. Gregorio Marañon, Madrid, Spain; 3University Medical Center Utrecht, Utrecht, The Netherlands; 4University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5Cellex, Dresden, Germany; 6ICREA, Barcelona, Spain.
Maria Salgado1, M Kwon2, M Nijhuis3, J van Lunzen4, Julià Blanco1, J Schulze zur Wiesch4, G Hutter5, AM Wensing3, JL Diez2, Javier Martinez-Picado1,6 for the EpiStem Consortium
Results
Conclusions
Findings
Methods
Herein we have compared two EpiStem pa-tients with >20 months of follow up after transplantation. We observed how varia-tions in the transplantation proccess results in important di�erences in reservoir size.
Patient 1 (Pt1) and Patient 3 (Pt3) were transplanted in Hospital Gregorio Marañón, Madrid, Spain. Samples were shipped to irsiCaixa (Barcelona), UMC Utrecht and UMC Hamburg-Eppendorf and analyzed for reservoir and immunological studies.
- qVOA: 90 and120 Million bulk CD4+ T cells were used for the qVOA of patient 1 and 3 respectively.
- HIV DNA was quanti�ed using ddPCR in: -CD4+ T cells from peripheral blood leukapheresis -CD3+ T cells from Bone Marrow -CD4+ Cells from Ileum biopsy sorted using FACSaria II (Figure 4)
- Semiquantitative PCR was performed using Gag prim-ers with limited dilutions of CD4 T cells DNA.
- Susceptibility assay: CD4+ T cells from the patient were infected in vitro with R5 and X4 tropic viruses.
- Ultrasensitive viremia: 9ml of plasma and 2.5ml of CSF were ultracentrifuged and HIV-RNA quanti�ed using Abbott VL platform.
- Chimerism was performed by short tandem repeat PCR (STR-PCR).
- Antibody titers were determined by ELISA
- Activation markers (CD38 and HLA-DR) were deter-mined in CD4+ and CD8+ T-cells. Acknowledgements
QVOA
Pre-SCT Post-SCT
10-3
10-2
10-1
100
101
IUPM
(CD
4 T
cells
)
Ultrasensitive Viremia (Blood)
Pre-SCT Post-SCT
10-1
100
101
102
103
Tota
l HIV
-1 R
NA
(cop
ies/
ml)
Reservoir in GALT
Pre-SCT Post-SCT
10-1
100
101
102
103
104
105
Tota
l HIV
-1 D
NA
(cel
ls/m
illio
n CD
4+)
Reservoir in Blood
Pre-SCT Post-SCT
10-1
100
101
102
103
104
105
Tota
l HIV
-1 D
NA
(cel
ls/m
illio
n CD
4+)
EpiStem:Observational
study
Immunology
ClinicalHematology
BM and CB Donor Testing
Evaluation and Counceling
Virology
To date, Berlin patient's case provides the only evidence of long-term HIV-1 control after allogeneic stem cell transplantation (SCT), potentially due to the CCR5Δ32/Δ32 donor genotype. The case of the two Boston patients, without resulting in HIV cure, showed that the SCT procedure itself involves a tremendous reduction of the viral reservoir. The reason why this happened is unraveled yet.
EpiStem is a collaborative project to guide and investigate the potential for HIV cure in HIV-infected patients requiring allogeneic stem cell transplantation for hematological disorders. The EPISTEM consortium is composed of an expert panel of hematologists with experience in allogeneic SCT procedures along with infectious disease specialists, virologist and immunologists with expertise in the �eld of HIV-1, reservoirs and cure (Figure 1).
Principal achievements of the EpiStem Consortium:
1. EpiStem is the largest international cohort of allogeneic SCT recipients with HIV-1 infection currently including 14 transplanted patients (Figure 2), 4 of them with CCR5Δ32/Δ32 donor .
2. Longitudinal samples from peripher-al blood (phlebotomy and leukapheresis), gut biopsy, cerebrospinal �uid (CSF), lymph node and bone marrow have been collected and analyzed.
3. In parallel, 30000 cord blood units have been tested for CCR5Δ32/Δ32 muta-tion in Europe. 200 mutated units have been identi�ed and are available for new transplantations
4. Preliminary analysis of the cohort sug-gests that allogeneic stem cell transplan-tation resulted in a decay of the latent res-ervoir in all analyzed cases. (Figure 3).
Figure 1. EpiStem Consortium
Figure 2. EpiStem Patients
Figure 3. HIV reservoir after SCT
NK-NHLHIV-1+ X4 tropic
Patient 3
HLA-matched sibling (10/10)
2013
- Full Chimera (30 days)- GvH disease
+20 month post SCTReduced intensity
conditioning
Peripheral Blood Progenitor Cells SCT
CCR5 wt
- no cART interruption
Patient 3
- no cART interruption
Myeloablative Haplo-cord SCT CCR5 wt
Burkitt NHLHIV-1+ R5 tropic
Patient 1
Cord Blood(5/6) CD34+ HLA-mismatched
donor
2012
- Chimera 0.2% BM/ 0.1% PB- No GvH disease
+29 month post SCT
Patient 1
- qVOA (IUPM) - Total DNA(cop/106 CD4) - Susceptibility(R5 and X4 viruses) - Immune supression - Activation Markers (HLA-DR+ CD38+ cells)
- SCA(HIV-RNA cop/ml)
- Ab titers
- Ileum CD4+ cells
- BM CD3+ cells
- CSF
CD4+ T cells
Plasma
Tissue
Allogeneic SCT with Peripheral Blood Progenitor Cells of an HLA-matched sibling donor, with a faster achievement of full donor chimera and a chronic GvH disease resulted in a more drastic reduction of the latent reservoir down to undetectable levels. We hypothesize that the “graft versus HIV-1 reservoir e�ect” contributes to facilitate the clearance of the viral reservoir.
We would like to thank all the collaborators, doctors, clinical sta� and researchers from the EpiStem Consortium hospitals and research centers that have participated somehow to the sample collection and processing (listed at www.epistem-project.org). We also thank to the founders: amfAR (Arche program) and The Spanish ministry of Economy
CD45 FITC CD3 APC
CD8
Perc
P
Figure 4. Ileum sorting strategy
0.034
25
Susceptible
69% of CD8+ inhibition
CD4: 11.30%CD8: 19%
5
Low levels
N/A
N/A
N/A
Undetectable
Undetectable
Susceptible
Non E�ective CD8+ T response
CD4: 1.41%CD8: 7.3%
Undetectable
Very Low levels
DNA Undetectable
DNA Undetectable
RNA Undetectable
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6 0
Pt 7
Pt 8
Pt 10
Pt 11
Blood
Apheresis
Ileum biopsiesCSF
Bone MarrowPt 12
Months post-transplantation5 10 15 2520 30 35
Pt 1760
Pt 9
Pt 20
CCR5WT/WT
CCR5∆32/∆32
Adult Donor 9* 2 11
Umbilical cord 1 2 3
10 4*CCR5 ∆32/WT
CD4 cells
