Understanding the potential

mechanisms of HIV cure by stem cell

transplantation

Annemarie Wensing, MD, PhD

University Medical Center Utrecht

WITS RHI University of the Witwatersrand Johannesburg

• HIV-DNA persists as viral reservoir

• If ART is interrupted HIV rebounds from this reservoir

Besson, G. J. et al. Clin. Infect. Dis. 2014

HIV-DNA remains despite ART

https://www.semanticscholar.org/paper/Are-T-cells-the-only-HIV-1-reservoir-Kandathil-Sugawara

HIV-RNA = viral load after start ART

HIV-DNA after start ART

Time (days) Time (years)

Proviral DNA in CD4 T-cells

CD4 T-cells

Where is this reservoir?

The reservoir is not only present in blood

but throughout the body

gp41

gp120

V3 loop

CD4 Binding

CD4

Co-receptor Binding

CD4 positive human cell

• HIV uses two human receptors to invade cells: CD4 and a co-receptor

Different co-receptors are used by HIV for

entry

CCR5

Hartley et al. Aids Res Hum Retroviruses 2005;21(2):171-89

CXCR4

R5

X4

Dual

Early in infection uses HIV the CCR5 receptor for entry = R5 troop

Late in infection HIV may start to use the CXCR4 receptor for entry = X4 troop

Cure Strategies

To limit the

establishment of the

reservoir

To reduce the size of

the reservoir

To control the

reservoir

Early

ART

Render uninfected

cells resistant to

HIV

Deplete infected

cells

Flush out the latent reservoir

Vaccine Immuno-therapy

Measuring the reservoir

Inspired by Nicholos Chomont. HIV reservoirs and strategies towards an HIV cure. HIV-NAT 2017

Markers of persistence Single Copy Assay: an extremely sensitive method that can even measures one copy of HIV-RNA at a time. Input: Plasma

https://www.projectinform.org/

PCR 2-LTR circles: ongoing replication qPCR/ddPCR can measure cell-

associated HIV RNA (caRNA) or total HIV DNA but does not discriminate between replication competent and defective HIV.

The Tat/Rev Inducible Limiting Dilution Assay (TILDA) is a polymerase-chain-reaction-based (PCR-based) assay that measures intracellular HIV RNA, no full distinction between replication competent variants or not

Bruner et al. Nature 2016

>95% of the measured HIV DNA is defective and

does not reflect replication competent virus

Markers of persistence

https://www.projectinform.org/

PCR 2-LTR circles: ongoing replication

Quantitative viral outgrowth assay (QVOA) is meant to measure replication-competent latent provirus. Input: CD4+ T cells retrieved by leukapheresis.

The Tat/Rev Inducible Limiting Dilution Assay (TILDA) is a polymerase-chain-reaction-based (PCR-based) assay that measures intracellular HIV RNA, no full distinction between replication competent variants or not

Applied techniques may overestimate or

underestimate the HIV reservoir

Challenges in qualifying the reservoir

• Only a small amount of CD4 T cells in PLWH on ART harbor HIV

• Intact or defect provirus

• Compartimentalization

• Which successes do we know?

Patient no more: The Berlin Patient

Stem cell transplantation procedure

https://www.miltenyibiotec.com/BE-en/products/cell-therapy/clinimacs-cd34-reagent-system-fda-approved/Patients/what-is-allogeneic-stem-cell-transplantation.html

= 100% chimera

Berlin Patient follow up

Gero Hütter et al. NEJM 2009

Stop HAART

18

wt/wt wt/32 32/32

“Resistant” to HIV infection

Normal R5 receptor

Heterozygote: less CCR5

Homozygote gene defect: no CCR5

Liu R, et al. Cell. 1996;86:367-367. Samson M, et al. Nature. 1996;382:722-725. Dean M, et al. Science. 1996;273:1856-1862. Huang Y, et al. Nat Med.1996;2:1240-1243. Michael NL, et al. Nat Med. 1997;3:1160-1162. Eugen-Olsen J, et al. AIDS. 1997;11:305-310.

1-2% of white people (Caucasians) do have a gene defect (Δ32 DNA) IN these people the CCR5 receptor is not present at the cell surface

Timothy Brown received stem cells with a

gene defect

How was Timothy Brown cured?

• Use of donor cells with genetic defect: CCR5Δ3232

• Absence of viruses able to use other co-receptors (i.e. X4 tropic-virus)

But

• Heavy Immune suppressive treatment (i.e. ATG levels)

• Total body irradiation

• Graft Versus Host Disease

• Two Stam Cell Transplantations

• Patient’s own CCR5 heterozygous state (smaller HIV-DNA reservoir?)

-

Gero Hütter et al. NEJM 2009

• Does this work for all patients who receive a SCT?

The Essen patient

Kordelas et al. N Engl J Med 2014 , Verheyen CID 2018.

• ART was stopped before SCT

• Treatment interruption led to rapid rebound of X4 tropic virus

Verheyen et al. CID, 2018

• clearly two distinct viral populations • 100% of the viral sequences detected after SCT were predicted to be X4-tropic • dominant X4-tropic viral sequence observed after SCT -present prior to SCT (4.4% of proviral DNA population) -present prior to treatment interuption

-287d/RNA/V03 -103d/DNA/V02

-103d/DNA/V11 -103d/RNA/V03 -103d/RNA/V04 -103d/RNA/V01 -103d/RNA/V05 -103d/RNA/V02

-18d/DNA/V05 -103d/DNA/V04

-103d/DNA/V10 -287d/RNA/V01 -103d/DNA/V01

-103d/DNA/V08 -18d/DNA/V06

-18d/DNA/V04 -18d/DNA/V02

-18d/DNA/V03 -287d/RNA/V02 -18d/DNA/V01

-103d/DNA/V03 -103d/DNA/V09

-18d/DNA/V07 -103d/DNA/V07

-103d/DNA/V06 -287d/RNA/V04

+373d/RNA/V06 +20d/RNA/V01 +373d/RNA/V02 -103d/DNA/V05 +373d/DNA/V01

+373d/RNA/V03 +373d/RNA/V08 +20d/RNA/V02 +373d/RNA/V01 +373d/RNA/V05 +373d/DNA/V02 +373d/DNA/V03 +373d/RNA/V04 +373d/RNA/V07

0.01

The Essen patient

Henrich, et al. Ann. Intern. Med. 2014

• Boston patients were transplanted with wildtype CCR5 donor cells

• Despite drastic reduction of the reservoir, HIV was able to rebound after ATI at 12 cq 32 weeks

The Boston patients

International collaboration to guide and investigate the potential for HIV cure in HIV-infected patients requiring allogeneic stem cell transplantation for hematological disorders

AIM 1 To guide clinicians involved in allogeneic SCT procedures in HIV infected individuals

AIM 2 To better understand the underlying biological processes leading to viral reservoir reduction and potential cases of HIV-1 eradication/remission.

www.icistem.org

Principal Investigators:

Javier Martinez Picado

Annemarie Wensing

Registration overview

Analyses of the dynamics of the viral reservoir

These unpublished data are not available at the website. For more information: See Salgado et al. https://www.annals.org/article.aspx?doi=10.7326/M18-0759

• How do we find out whether these patients has been

cured?

IciStem #19 The “Duesseldorf patient”

Stem cell transplantation with CCR5Δ32 Homozygous Allo-HSCT

ATI Since November 2018 without viral rebound

120

100

80

60

40

20

0

900 700 500 300 100 0

Jensen et al. CROI 2019.

• No proviral DNA detectable in blood, bone marrow, lymph nodes, liqour and rectum

• DNA and RNA scope positive (Jake Estes) • Western blot fading (antibodies)

IciStem #19 The “Duesseldorf patient”

Jensen et al. CROI 2019.

• DNA and RNA scope positive (Jake Estes)

IciStem #19 The “Duesseldorf patient”

IciStem #36 The “London patient”

Days post allo-HSCT

Gupta et al. Nature 2019

Stem cell transplantation with CCR5Δ32 Homozygous Allo-HSCT

Gupta et al. Nature 2019

IciStem #36 The “London patient”

CCR5 genotyping Pre-SCT: wildtype

Post-SCT: CCR5∆32

Immune responses post-SCT

Time (days) Gupta et al. Nature 2019

IciStem #36 The “London patient”

Overview CCR5∆32 homozygous HSCT

transplants

“Berlin patient” “London patient” “Duesseldorf patient”

CCR5∆ 32 heterozygous WT CCR5 homozygous WT CCR5 homozygous

R5 tropic HIV infection R5 tropic HIV infection R5 tropic HIV infection

Acute myeloid leukemia Hodgekin lymphoma Acute myeloid leukemia

2 HSCTs Single HSCT Single HSCT

Total body irradiation No irradiation No irradiation

Full intensity conditioning Reduced intensity

conditioning

Reduced intensity

conditioning

Mild GvHD (skin) Mild GvHD (skin/gut) Moderate GvHD (liver)

100% T-cell donor

chimerism

100% T-cell donor

chimerism

100% T-cell donor

chimerism

• Allogeneic Stem cell transplantation has a high mortality risk

• Only suitable for patients with a severe haematological malignancy

• Chances to find an adult matching donor for non Caucasian patients are extremely low

Is this approach safe and scalable?

Summary

• IciStem has identified > 40.000 CCR5Δ32 donors that can be used for allo-SCT also IciStem has compiled the largest registry of allo-transplants in people living with HIV

• After allo-SCT, a sharp decline in HIV DNA in the blood and

tissues is observed to below the level of detection in most transplanted patients

• Important determinants for cure after allo-SCT are: single

transplant CCR5Δ32 donor cells, no TBI, no full intensity chemo,

• The case of the Berlin Patient was not a miracle

• “Everything that happens once can never happen again. But everything that happens twice will surely happen a third time.” (Paulo Coelho)

IciStem research workgroup: Julià Blanco (Immunologist, AIDS Research Institute IrsiCaixa, Barcelona), Jorge Carrillo (Immunologist, AIDS Research Institute IrsiCaixa, Barcelona), Johanna Eberhard (Immunologist, University Medical Center Hamburg-Eppendorf), Mi Kwon (Hematologist, Hospital Gregorio Marañón), María Salgado (Virologist, AIDS Research Institute IrsiCaixa, Barcelona) Management team: Tineke Johnston and Josien Straver (Financial management/project assistance University Medical Center Utrecht), Laura Huyveneers (Medical coordination/IRB University Medical Center Utrecht), Pascual Balsalobre (Gregorio Marañón, Madrid), Judith Dalmau (AIDS Research Institute IrsiCaixa, Barcelona) Scientific Advisors: Koen van Besien (New York-Presbyterian hospital) IciStem participants Belgium: Linos Vandekerckhove, Marie-Angélique de Scheerder, Eva Steel (University of Ghent) Canada: Lisa Barrett, Sharon Oldford, Jill Moore, Clarissa Brisseau (NSHA/Dalhousie University, Halifax) Germany: Dieter Häussinger, Guido Kobbe, Björn Jensen (University Hospital Düsseldorf), Rolf Kaiser, Elena Knops (University of Cologne) Maximilian Christopeit (University Medical Center Hamburg-Eppendorf)

Italy: Alessandra Bandera, Antonio Muscatello and Alessandro Soria (San Gerardo Hospital, Monza, Italy), Gabriella Scarlatti, Simona Piemontese (Istituto Scientifico San Raffaele) Netherlands: Pauline Ellerbroek, Lodewijk Brosens, Anke Bruns, Erik van Maarseveen (UMC Utrecht) Jan van der Meer, Sacha Zeerleder (AMC), Jaap Jan Boelens (University Medical Center Utrecht/Memorial Sloan Kettering Cancer Center, New York) Spain: Jon Badiola, Manuel Jurado Chacón (Complejo Hospitalario Universitario de Granada), Raquel Saldana (Hospital General de Jerez de la Frontera), Luz Martín Carbonero (La Paz Hospital), Ildefonso Espigado (University Hospital Virgen del Rocío, Seville) Sweden Piotr Nowak (Karolinska Institutet) Switzerland Mitja Nabergoj, Alexandra Calmy (Hôpitaux Universitaires de Genève) United Kingdom: Kavita Raj, Fabio Cruciani, Varun Mehra, Carmel Rice (Kings College Hospital, London) Angela Bailey (Imperial College Healthcare NHS Trust, London), Waseem Qasim (Institute of Child Health & Great Ormond Street Hospital, London), Ravindra Gupta (University College London Hospital)

Acknowledgements The IciStem consortium

Javier Martinez-Picado (Co-PI,

Virologist, AIDS Research Institute IrsiCaixa, Barcelona)

Annemarie Wensing (Co-PI, Clinical

Virologist, University Medical Center Utrecht)

Jose L. Díez Martin (Hematologist,

Hospital Gregorio Marañón, Madrid)

Gero Hütter (Hematologist, Cellex

Dresden) Jürgen Kuball (Hematologist, University

Medical Center Utrecht)

Monique Nijhuis (Virologist,

University Medical Center Utrecht)

Vanderson Rocha (Hematologist

Cord Blood Bank Specialist Oxford University)

Asier Sáez-Cirión (Immunologist,Pasteur Institute, Paris) Julian Schulze zur Wiesch (Infectious disease specialist, UMC Hamburg-Eppendorf)

Translational Virology, UMC Utrecht