Long-Term Reduction in Peripheral Blood HIV-1 Reservoirs Following Reduced-

Intensity Allogeneic Stem Cell Transplantation in Two HIV-Infected

IndividualsTimothy J. Henrich1,2, Gaia Sciaranghella3, Jonathan Z. Li1,2, Sebastien

Gallien4, Vincent Ho5,2, Ann S. LaCasce5,2, and Daniel R. Kuritzkes1,2

1Brigham and Women's Hospital, Boston, MA; 2Harvard Medical School, Boston, MA; 3Ragon Institute of MGH, MIT, and Harvard, Boston, MA; 4 Hopital Saint-Louis, Paris, France;

5Dana-Farber Cancer Institute, Boston, MA.

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Background• One reported “functional cure” of HIV-1 infection: myeloablative

allogeneic HSCT from a homozygous ccr5Δ32 donor1,2

• Several factors may have contributed to functional cure including pre-transplant myeloablative chemotherapy, GVHD, full engraftment of CCR5- donor cells

• Cytotoxic chemotherapy alone insufficient to eliminate reservoirs as HIV-1 DNA persists after autologous HSCT3,4

• The long-term effects of allogeneic HSCT using CCR5+ stem cells have not been studied in detail

1Hutter et al. 2009; 2Allers et al. 2010;3Simonelli et al. 2010; 4Cillo et al. 2011;

Study AimsStudy Aims:Examine long-term changes in the peripheral HIV-1 reservoir

following allogeneic HSCT in the setting of cARTExplore HIV-1 coreceptor usage, PBMC coreceptor

expression and HIV-specific antibody responses pre- and post-HSCT

Patients:2 HIV-1 infected patients on combination ART who underwent reduced-intensity conditioning (RIC) allogeneic HSCTRIC = non-myeloablative chemotherapy, no total body irradiation or anti-thymocyte globulin

MethodsStudied stored blood samples collected pre- and post-HSCT and

prospectively collected samples (5 time points)

1) Quantified proviral HIV-1 DNA from peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells by real-time PCR

2) Quantified 2-LTR circles from PBMC episomal DNA

3) Quantified plasma viremia by a single-copy assay

4) Viral outgrowth assays using ~107 patient-derived CD4+ T cells and CD8 T cell-depleted lymphoblasts from an HIV-negative donor

5) CCR5 genotyping/flow cytometric quantification of CCR5 expression on CD3+ T lymphocytes

6) Genotypic and phenotypic determination of HIV-1 coreceptor usage

7) Quantified HIV-1-specific Ab levels & avidity

Study Patients Patient A:

Male with perinatally acquired HIV-1 on long-term ART 2006: Stage IV Hodgkin disease standard treatment Disease recurrence salvage therapy 2007: Autologous HSCT 2008: Relapse RIC partially mismatched unrelated-donor HSCT

cART: TDF/FTC/EFV 3-4 years pre-HSCT with undetectable VL

Clinical course post-allogeneic HSCT

Study Patients Patient B:

Male with sexually acquired HIV-1 in mid-1980’s 2003: Large B-cell lymphoma chemotherapy and cART started 2006: New stage IV Hodgkin lymphoma

Disease recurrence salvage therapy 2007: Autologous HSCT 2010: MDS (Tx-related) RIC matched related-donor HSCT

cART: TDF/FTC/RAL peri-transplant with undetectable VL

Clinical course post-allogeneic HSCT

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

50100150200250300

HIV-

1DN

A(c

opie

s/106

PBM

C)

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

200400600800

1000

Days afterHSCT

CD4+

TCe

lls(p

erm

m3))

100% donor lymphochyte

chimerism

<1.8

TND

65 TND

TND

104

VL (clinical lab)

VL (SCA)

<0.5<1.8

<1.8

Patient A

Viral outgrowth assay negative day +1266

No 2-LTRsdetected

Patient B

DLI= donor lymphocyte infusion

0 100 200 300 400 500 600 7000

100

200

300

HIV-

1DNA

(cop

ies/1

06PB

MC)

0 100 200 300 400 500 600 7000

200400600800

1000

Days afterHSCT

CD4+

TCe

lls(p

ermm

3 )

VL (clinical lab)

VL (SCA)

<1.8

TND

TND <4

8

100% donor granulocyte chimerism

DLI

<0.5<1.8

<1.8

Viral outgrowth assay negative day +652

No 2-LTRsdetected

CCR5 / Coreceptor Usage• Both patients heterozygous for ccr5Δ32 mutation• PBMC homozygous wild-type for CCR5 after engraftment• Percentage of CCR5-expressing lymphocytes nearly doubled

after full donor engraftment in Patient A (sufficient sample)

• Full-length HIV-1 env amplified from proviral DNA at pre- and 1st post-HSCT PBMC samples (later timepoints negative)

• V3-loop genotyping predicted CCR5 usage pre- and post-HSCT• R5 phenotype confirmed by tropism assay of pseudotyped viruses

expressing PBMC-derived env

Anti-HIV Ab Quantification

0 200 400 600 800 1000 12000123456789

1011

Subject ASubject B

Day Post-HSCT

HIV-

1 A

b L

evel

(S/C

)

0 200 400 600 800 1000 12000

1

2

3

4

Subject ASubject B

Day Post-HSCTLA

g-Av

idity

OD

n• HIV-specific Ab detected by VITROS assay pre- & post-HSCT• Decrease in Ab levels post-HSCT from diluted and undiluted

plasma• Similar decrease in antigen avidity by limiting-antigen assay

Limited Sensitivity VITROS Assay Limiting-Antigen Avidiy Assay

Patient APatient B

Summary & Conclusions• Allogeneic HSCT with RIC in the setting of suppressive ART led

to a substantial and sustained reduction in the HIV-1 reservoir in PBMC

- Reduction in proviral HIV-1 DNA correlated temporally with full donor engraftment

• Engraftment of susceptible donor cells without infection adds supportive evidence that HIV-1 replication is fully suppressed by effective cART

• Declining HIV-specific Ab levels/avidity provide further evidence for minimal persistence of HIV-1 antigen

• Tissue sampling and analytic treatment interruption are necessary to fully assess the extent of HIV-1 reservoir reduction after allogeneic HSCT

AcknowledgementsBWH: Funding Sources:Members of the Kuritzkes Lab: NIH/NIAID 1K23AI098480-01A1 to TJHZixin Hu UM1 AI068636 to DRK; P30 AI060354 Nina Lin U19 AI096109 to SGDFrançoise GiguelLaura LavoieAthe TsibrisMembers of the ID Division

Blood Systems Research Institute/UCSF:Michael BuschSheala KeetingMila Lebedeva

UCSF:Steven Deeks

Harvard School of Public Health:Ronald Bosch (SDAC)

Viral DNA Diversity

Pre-HSCTPost-HSCT

Patient ASingle genome sequencing of full-length HIV-1 Env from PBMC DNA from pre- and 1st post-HSCT samples

No major evolutionary changes observed following HSCT