Autopsy Study Defines Composition and Dynamics of the HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell Transplantation with CCR5Δ32/Δ32 Donor Cells

Abstract

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

Full text

Citation: Huyveneers, L.E.P.; Bruns,
A.; Stam, A.; Ellerbroek, P.; de Jong,
D.; Nagy, N.A.; Gumbs, S.B.H.;
Tesselaar, K.; Bosman, K.; Salgado,
M.; et al. Autopsy Study Defines
Composition and Dynamics of the
HIV-1 Reservoir after Allogeneic
Hematopoietic Stem Cell
Transplantation with CCR5∆32/∆32
Donor Cells. Viruses 2022,14, 2069.
https://doi.org/10.3390/v14092069
Academic Editor: Sonia Moretti
Received: 30 August 2022
Accepted: 14 September 2022
Published: 17 September 2022
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
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4.0/).
viruses
Article
Autopsy Study Defines Composition and Dynamics of the
HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell
Transplantation with CCR5∆32/∆32 Donor Cells
Laura E. P. Huyveneers 1, Anke Bruns 2,3, Arjen Stam 1, Pauline Ellerbroek 2, Dorien de Jong 1, Noémi A. Nagy 1,
Stephanie B. H. Gumbs 1, Kiki Tesselaar 4, Kobus Bosman 1, Maria Salgado 5,6 , Gero Hütter 7, Lodewijk A.
A. Brosens 8, Mi Kwon 9, Jose Diez Martin 9, Jan T. M. van der Meer 10 , Theun M. de Kort 11, Asier Sáez-Cirión12 ,
Julian Schulze zur Wiesch 13 , Jaap Jan Boelens 14,15 , Javier Martinez-Picado 5,6,16,17 , Jürgen H. E. Kuball 3,4 ,
Annemarie M. J. Wensing 1,*,† and Monique Nijhuis 1,† ,‡ on behalf of the IciStem Consortium
1Department of Medical Microbiology, University Medical Center Utrecht (UMCU),
3584 CX Utrecht, The Netherlands
2Department of Internal Medicine and Infectious Diseases, University Medical Center Utrecht (UMCU),
3584 CX Utrecht, The Netherlands
3
Department of Hematology, University Medical Center Utrecht (UMCU), 3584 CX Utrecht, The Netherlands
4Central Laboratory of Translational Immunology, University Medical Center Utrecht (UMCU),
3584 CX Utrecht, The Netherlands
5IrsiCaixa AIDS Research Institute and Institute for Health Science Research Germans Trias i Pujol (IGTP),
08916 Badalona, Spain
6
Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto
de Salud Carlos III, 28029 Madrid, Spain
7DKMS CC, 72072 Tübingen, Germany
8Department of Pathology, University Medical Center Utrecht (UMCU), 3584 CX Utrecht, The Netherlands
9Department of Hematology, Hospital General Universitario Gregorio Maranon, 28007 Madrid, Spain
10 Department of Internal Medicine, Amsterdam UMC, 1105 AZ Amsterdam, The Netherlands
11 Department of Anatomy and Embryology, Leiden University Medical Center,
2333 ZA Leiden, The Netherlands
12 HIV Inflammation and Persistence, Institut Pasteur, UniversitéParis Cité, 75015 Paris, France
13 Department of Internal Medicine, UMC Hamburg-Eppendorf, 20251 Hamburg, Germany
14 Department of Hematology and Oncology, Princess Maxima Center, 3584 CS Utrecht, The Netherlands
15 Stem Cell Transplant and Cellular Therapies, Pediatrics, Memorial Sloan Kettering Cancer Center,
New York, NY 10065, USA
16 Catalan Institution for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain
17 University of Vic—Central University of Catalonia (UVic—UCC), 08500 Vic, Spain
*Correspondence: a.m.j.wensing@umcutrecht.nl
† These authors contributed equally to this work.
‡ Study Group team members are listed in the Acknowledgments.
Abstract:
Allo-HSCT with CCR5
∆
32/
∆
32 donor cells is the only curative HIV-1 intervention. We
investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in
two patients. IciS-05 and IciS-11 both received a CCR5
∆
32/
∆
32 allo-HSCT. Before allo-HSCT, ul-
trasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis;
deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ul-
trasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep
sequencing, and viral characterization were done in post-mortem tissue samples. Both patients har-
bored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture.
Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell
subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-
mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient
IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death,
whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5
∆
32/
∆
32,
allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to
those present before transplantation persisted in post-mortem-obtained tissues, indicating that these
tissues play an important role as viral reservoirs.
Viruses 2022,14, 2069. https://doi.org/10.3390/v14092069 https://www.mdpi.com/journal/viruses

Viruses 2022,14, 2069 2 of 12
Keywords: HIV-1; HIV persistence; reservoir; cure; tissue; CCR5∆32; allo-HSCT
1. Introduction
Although antiretroviral therapy (ART) successfully averts HIV-1-related disease pro-
gression and saves lives of millions of HIV-infected individuals, the ongoing inflammation
and costs urgently call for curative strategies [
1
]. Current antiretroviral compounds target
several steps in the viral life cycle but do not target the integrated provirus nor suppress
HIV-1 transcription and production from the cellular reservoir. This integrated provirus
forms a stable viral reservoir and is the major barrier to HIV-1 eradication [1].
Several approaches are being explored to eliminate the viral reservoir and ultimately
convert HIV-1 infection into a curable disease. Successful viral remission is known in four
cases of treatment interruption after allogeneic hematopoietic stem cell transplantation
(allo-HSCT) with homozygous CCR5
∆
32 donor cells: “The Berlin patient”, two partici-
pants in the IciStem cohort; IciS-36, IciS-19—also named “The London patient” and “The
Düsseldorf patient”; and recently in a woman of mixed race in New York. The cells used
for transplantation lack expression of surface CCR5, rendering them resistant to infection
by CCR5-tropic HIV-1 [
2
–
7
]. Though no viral rebound was observed in these patients
after treatment interruption (ATI), traces of proviral HIV-1-DNA could still be found in
some samples [
3
,
5
,
7
,
8
]. Also, in patients transplanted with CCR5-expressing donor cells
(CCR5WT), a profound reduction in the viral reservoir was observed. However, in these
patients, therapy interruption resulted in a delayed rebound of viruses comparable to
the pre-allo-HSCT PBMC population in “the Boston patients”, but not in “the Minnesota
patient” [9–12].
These data highlight the knowledge gap on how allo-HSCT impacts the viral reservoir
and which cellular or anatomical compartments fuel HIV-1 rebound. IciStem is an interna-
tional collaboration to guide and investigate the potential for HIV-1 cure after allo-HSCT
and has compiled the largest cohort of HIV-1-infected individuals receiving allo-HSCT
(www.icistem.org, accessed on 15 August 2022). Two IciStem CCR5
∆
32 patients (IciS-05
and IciS-11) died after allo-HSCT. Post-mortem biopsies of these patients provided a unique
opportunity to broadly investigate the viral reservoir after allo-HSCT in multiple tissues,
which would never be feasible in living patients.
1.1. Cases
1.1.1. Patient IciS-05
A 38-year-old male presented in 1998 with a CD4
+
T-cell count of 46 cells/
µ
L whole
blood and an HIV-1 viral load (VL) >500,000 RNA copies/mL plasma. Despite several
changes in his therapy regimen, full suppression was not reached until 2008 (Table 1). Ex-
tensive drug resistance was selected and genotypic analyses of the viral envelope predicted
infection with CCR5-tropic virus. In 2011, poor-risk myelodysplastic syndrome (MDS)
was diagnosed, necessitating allo-HSCT in May 2012. Because no adult HLA-matched
unrelated CCR5
∆
32 donor was found, a cord-blood transplant of a homozygous CCR5
∆
32
donor (HLA-match 4/6) combined with an infusion of CD34+-cells of an HLA-unmatched
haploidentical family donor (CCR5wt) was performed, to aim for HIV-1 cure but also
to ensure faster engraftment (Table 1). Using general diagnostic assays, viral load was
20 copies/mL on the day of transplant and remained suppressed (<50 copies/mL) after
transplantation. Cord-blood donor chimerism in peripheral blood lymphocytes (PBLs) was
57% on day +16 and 100% on day +36. On day +54, chimerism reduced to 95%, and further
down to 85% at day +65. Unfortunately, the patient died of severe sepsis on day +68.

Viruses 2022,14, 2069 3 of 12
Table 1. Baseline clinical characteristics of patients.
IciS-05 IciS-11
Hematological Data
Hematological diagnosis MDS AML
Donor type/graft source Cord blood + CD34+-cells third party donor HLA-matched unrelated donor
Donor CCR5 Homozygous CCR5∆32 First donor: homozygous CCR5∆32, second donor:
heterozygous CCR5∆32/WT
Recipient HLA
HLA-A*03:01;24:02;
HLA-B*07:02;35:01;
HLA-Cw*04:01;07:02; HLA-DRB1*01:01;04:04;
HLA-DQB1*03:02;05:01
HLA-A*01:01;02:01;
HLA-B*07:02;-;
HLA-Cw*07:02;-;
HLA-DRB1*15:01;-;
HLA-DQB1*06:02; -
Donor-recipient HLA match
4/6 cord blood donor (A*03:01; -; B*07:02; 35:02;
C*07:02; 04;01;
DRB1*01:01; 11:04);
50% haploidentical family member
(A*24:02;26:01;B*07:02; 14:01;C*07:02;08:02;
DRB1*04:04; 14:54; DQB1*03:02; 05:03)
10/10; 10/10
Pre-transplant chemotherapy None 2 induction cycles with cytarabine and idarubicin
Conditioning ATG, fludarabine with busulvex
ATG, fludarabine, and low-dose TBI before initial
transplant; ATG fludarabine and treosulfan before
second transplant
GvHD prophylaxis Cyclosporine, prednisone Cyclosporine, prednisone, and mycophenolic acid
mofetil
GvHD severity Acute skin GvHD grade 1 No GvHD
Virological data
Time from HIV-1 diagnosis to allo-HSCT 14 years 22 years
Time from start cART to allo-HSCT 14 years 19 years
Host CCR5 CCR5WT/WT CCR5WT/WT
Predicted HIV-1 co-receptor tropism CCR5-tropic virus (FPR 68.0–96.2%) CCR5-tropic virus (FPR 9.7–77.3%)
Phenotypic HIV-1 co-receptor tropism CCR5-tropic virus CCR5-tropic virus
HIV-1 subtype HIV-1 subtype B HIV-1 subtype B
cART History
1998: AZT/3TC, NFV. 2005: TNF, EFV, ATV/r 2006:
TNF, LPV/r, NVP 2006: TNF, SQV/r, NVP
2008: AZT/3TC, TNF, SQV/r
1996: AZT, DDI.
1996: D4T, 3TC, SQV
1997: D4T, 3TC, SQV/r
1999: D4T, 3TC, NVP
2003: TNF, 3TC, NVP
cART during allo-HSCT procedure
Day -152: TNF/FTC, DRV/r, RAL
Day -34 until +68: TNF/FTC, RAL, MVC, ENF
Day -5 until +37: ETR added
Day -19: TNF, FTC, DTG
Day +20 until +107: ABC/3TC, DTG
Plasma HIV RNA load at allo-HSCT 20 copies/ml <50/TND copies/ml
HCV Anti-HCV negative Anti-HCV negative
HBV HbsAg negative, anti-HBc positive, anti-Hbs
negative
HbsAg negative, anti-HBc positive, anti-Hbs
positive
CMV status pre-SCT Positive Positive
Donor CMV status Positive Positive
3TC, lamivudine. ABC, abacavir. Allo-HSCT, allogeneic hematopoietic stem cell transplantation. AML, acute
myeloid leukemia. Anti-HBc, antibody against hepatitis B core antigen. Anti-HBs, antibody against hepatitis B
surface antigen. ATG, anti-thymocyte globulin. ATV/r, ritonavir boosted atazanavir. AZT, zidovudine. cART,
combined antiretroviral therapy. CCR5, C-C chemokine receptor type 5. CCR5
∆
32, C-C chemokine receptor
type 5 delta 32 mutation. CMV, cytomegalovirus. DDI, didanosine. DRV/r, ritonavir boosted darunavir. DTG,
dolutegravir. D4T, stavudine. EBV, Epstein-Barr virus. EBV Vca, EBV viral capsid antigen antibody. EBV EBNA,
EBV nuclear antigen antibody. EFV, efavirenz. ENF, enfuvirtide. ETR, etravirine. FTC, emtricitabine. FPR, False
Positive Ratio. GvHD, Graft-versus-host disease. HBV, hepatitis B virus. HbsAG, hepatitis B surface antigen. HCV,
hepatitis C virus. HLA, human leukocyte antigen. LPV/r, ritonavir boosted lopinavir. MDS, myelodysplastic
syndrome. MVC, maraviroc. NFV, nelfivnavir. NVP, nevirapine. RAL, raltegravir. RS, respiratory syncytial virus.
RTV or r, ritonavir. SQV/r, ritonavir boosted saquinavir. TBI, total body irradiation. TDF, tenofovir disoproxil
fumarate. TND, target not detected. TPHA, Treponema pallidum haemagluttination test. WT, wild type.
1.1.2. Patient IciS-11
A 38-year-old male was diagnosed with HIV-1 in 1993 with a CD4+ T-cell count of
440 cells
/. In March 1996, when CD4+ T-cell count fell below 200 cells/
µ
L whole blood, the
patient started dual therapy, quickly followed by triple therapy (Table 1). In December 2014,

Viruses 2022,14, 2069 4 of 12
when VL was suppressed and CD4+ T-cells were around 700 cells/
µ
L, he was diagnosed
with poor-risk AML. Genotypic analyses of the viral envelope predicted infection with
CCR5-tropic virus.
In April 2015, the patient received allo-HSCT with cells of a 10/10 HLA-matched
unrelated donor homozygous for the CCR5
∆
32 mutation (Table 1). Before allo-HSCT, CD4
count was 283 cells/
µ
L and plasma VL remained suppressed. Donor chimerism decreased
from 60% to 0% between day +27 and +55. On day +41, donor chimerism in bone marrow
(BM) was only 18%, further indicating graft failure. On day +71, the patient received a
second transplant of a heterozygous CCR5
∆
32/WT unrelated donor (HLA-match 10/10);
the first donor was not available. A month after the second allo-HSCT, on day +100 after the
first allo-HSCT, full donor chimerism was reached. On day +108 after the first allo-HSCT
and day +37 after the second allo-HSCT, the patient died due to respiratory failure.
2. Materials and Methods
2.1. Patients and Patient Material
This study included two IciStem patients. IciS-05 was included after signing a general
waiver for the use of body material for future research, according to the regulations of
the UMC Utrecht HSCT Biobank (HSCT study number 3440). IciS-11 was included after
written informed consent was acquired. Ethical approval by the Institutional Medical
Ethics Committee of the UMCU was obtained under protocol number NL53114.041.15.
Before allo-HSCT, plasma, serum, PBMCs, and BM were obtained and a leukaphere-
sis was performed. From the leukapheresis, 2–3
×
10
8
PBMCs were sorted into T
N
(CD3
+
CD4
+
CD45RO
-
CCR7
+
CD27
+
Fas
-
), T
SCM
(CD3
+
CD4
+
CD45RO
-
CCR7
+
CD27
+
Fas
+
),
T
CM
(CD3
+
CD4
+
CD45RO
+
CCR7
+
CD27
+
), T
TM
(CD3
+
CD4
+
CD45RO
+
CCR7
-
CD27
+
Fas
+
)
and T
EM
(CD3
+
CD4
+
CD45RO
+
CCR7
-
CD27
-
Fas
+
). CD4
+
T-cells were obtained from base-
line PBMCs and (CD4
+
-cell Isolation Kit, negative selection) (Miltenyi Biotec). At different
time points after, allo-HSCT plasma and PBMCs were obtained. Post-mortem biopsies were
obtained the day after death from terminal ileum, lung, liver, and spleen. From patient
IciS-11, also brain biopsies were obtained and CD4
+
-cells were isolated from fresh ileum
and a mediastinal lymph node.
2.2. DNA Isolation
Total DNA was isolated from frozen tissue biopsies using two different methods
because of size diffence of the biopsy size and the maximum input in the the DNeasy
Blood & Tissue kit (Qiagen, Hilden, Germany). Biopsy 1 was sliced into small pieces and
cells were lysed using the MagNALyser (Roche, Basel, Switzerland) and purified using
QIAquick (Qiagen, Hilden, Germany). Biopsy 2 was lysed and purified using the DNeasy
Blood & Tissue kit (Qiagen, Hilden, Germany). Total DNA from PBMCs or CD4
+
-cells was
isolated using the DNeasy Blood & Tissue kit.
2.3. Patient and Donor CCR5 Genotype Determination
To determine the CCR5 genotype, 30 ng total cellular DNA was amplified using
CCR5forward2 5
0
-GATAGGTACCTGGCTGTCGTCCAT-3
0
, and CCR5d 5
0
-CCTGTGCCTCT
TCTTCTCATTTCG-3
0
. As a control, plasmids containing the wild type CCR5 sequence or
the CCR5
∆
32 sequence were also amplified and lengths of all amplified fragments were
compared by gel-electrophoresis and the Bioanalyzer (Agilent, Santa Clara, United States).
Deletions in CCR5 were confirmed by sequence analysis.
2.4. Ultra-Sensitive Viral Reservoir Quantification
Ultra-sensitive HIV-1 proviral DNA quantification was performed using primers in the
conserved HIV-LTR region and pol region and total cell DNA was quantified using an RPP30
(Ribonuclease P/MRP Subunit P30) primer and probe set as described in Bosman et al. [
13
].
HIV-1 DNA was quantified using de droplet digital PCR (ddPCR, Bio-Rad). The threshold
was set manually at a conservative fluorescence level. Water was used as a no-template

Viruses 2022,14, 2069 5 of 12
control and DNA isolated from PBMCs of HIV-negative donors was used as a DNA template
control. U1 cells were tested as a positive control. In 3/100 assays, a single positive droplet
was observed. Thus, measurement of a single droplet is interpreted as a trace.
2.5. Micro-Chimerism Assessment
In addition to routine clinical chimerism testing, ultra-sensitive chimerism PCR was
performed on peripheral blood (day +55, +106), post-mortem lymph node, and CD4
+
-
cells from the lymph node of patient IciS-11 using the Mentype DIPscreen and Mentype
DIPquant assays (Biotype), with a sensitivity of 0.01–0.001%, depending on quality and
quantity of the purified DNA.
2.6. Ultra-Sensitive Plasma HIV-1 RNA Determination
Ultra-sensitive VL was measured using Cobas Ampliprep/Cobas TaqMan HIV-1 Test
v2.0 (Roche Molecular Systems, Inc. Pleasanton, CA, USA) after ultracentrifugation of
4–9 mL of plasma [14].
2.7. HIV-1 Co-Receptor Prediction and Determination
Genotypic HIV-1 co-receptor tropism was predicted by deep sequence analysis of the
V3 loop of the viral envelope (gp160-V3). Total cellular DNA was amplified in triplicate in
a nested approach using primers envF1.1 5
0
-GGATATAATCAGYYTATGGGA-3
0
, envF1.2
5
0
-GAGGATATAATCAGTTTATGG
0
, envR1.1 5
0
-GGTGGGTGCTAYTCCYAITG-3
0
, envR1.2
5
0
-GGTGGGTGCTATTCCTAATGG-3
0
for the first amplification and primers envF2.1 5
0
-
GATCAAAGCCTAAARCCATGT-3
0
, envF2.2 5
0
-GATCAAAGCCTAAAGCCATG-3
0
, envR2.1
5
0
-CTCCAATTGTCCYTCATHTYTCC-3
0
, envR2.2 5
0
-ACTTCTCCAATTGTCCCTCATAT-3
0
in the nested amplification. The DNA concentration of the purified amplicons was deter-
mined using Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific, Waltham, MA,
USA) and a sequence library was formed using the Nextera DNA Library Preparation Kit
(Illumina, San Diego, CA, United States). Finally, the DNA concentration of the library was
measured (Quant-iT PicoGreen dsDNA Assay Kit, Thermo Fisher Scientific, MA, USA),
diluted to 2 ng/mm
3
denatured, and sequenced using the MiSeq Reagent Kit v2 (500-cycles)
(Illumina, San Diego, USA). Viral tropism was predicted in silico using geno2pheno (454)
with a 3.5% cut-off indicating the probability of classifying an R5-tropic virus falsely as
CXCR4-tropic virus (FPR; false-positive-rate) [15].
Phenotypic HIV-1 co-receptor was determined using cells and virus culture. Cells:
U373-MAGI-CCR5E, U373-MAGICXCR4CEM [
16
], MT2 cell lines [
17
] (NIH AIDS Reagent
Program), and SupT1R5X4 (personal gift from J. Hoxie) were maintained as recommended.
PBMCs from five healthy donors (homozygous for CCR5WT) were prepared by Ficoll–
Paque density gradient centrifugation of heparinized blood. The mix was stimulated
for
3 days
with phytohaemagglutinin (2 mg/L) in culture medium [RPMI1640 with L-
glutamine (BioWhittaker, Lonza, Basel, Switzerland), 10% fetal bovine serum (FBS; Biochrom
AG, Berlin, Germany) and 10 mg/L gentamicin (Gibco)]. Virus culture: PBMCs from
healthy donors were co-cultured with purified CD4
+
-cells from patients IciS-05 and IciS-11
for 2 h at 37
◦
C, after which cells were washed twice. Subsequently, cells were cultured with
25 U/mL IL-2 in 10 mL CM. Virus was cultured for 3 weeks, and twice-weekly half of the
culture was replaced with fresh culture medium with 25 U/mL recombinant human IL-2
(Thermo Fisher Scientific, Massachusetts, United States) and once weekly freshly stimulated
PBMCs from healthy donors were added. Twice weekly HIV-1 replication was monitored
by CA-p24 ELISA and culture supernatant was stored for analyses of viral co-receptor
tropism in MT2 cells (expressing CD4 and CXCR4) and as a control SupT1R5
×
4 cells
(expressing CD4, CCR5 and CXCR4).
Analysis of HIV-1 co-receptor usage in U373-MAGI cells: Infection of these cells was
done using 2 ng of CA-p24 of the patient-derived viral isolate or the control viruses [6].
Phylogenetic sequence analysis; viral evolution and compartmentalization: HIV-1
subtype was determined using REGA HIV Subtyping Tool [
18
]. The evolutionary history

Viruses 2022,14, 2069 6 of 12
was inferred using Maximum Likelihood (MEGA 7) (500 bootstraps) [
19
]. The tree with
the highest log likelihood is shown. Initial tree (s) for the heuristic search were obtained
automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood (MCL) approach, and then
selecting the topology with superior log likelihood value. The tree is drawn to scale, with
branch lengths measured in the number of substitutions per site. All positions containing
gaps and missing data were eliminated.
3. Results
3.1. Patient IciS-05
Twenty days before allo-HSCT, only an unquantifiably low amount of HIV-1 RNA could
be detected with routine diagnostics. Ultra-sensitive plasma viral load was
15 copies/mL
(Figure 1a). The viral reservoir was quantified in BM, PBMCs, naïve T-cells, and all memory
T-cell subsets with HIV-1-DNA being most abundant in the more differentiated T-cell subsets
(1135-6924 HIV-1-LTR copies/106cells) (Table 2).
Table 2. Results of HIV-1-DNA quantification and characterization in patient IciS-05.
Time
Point
(Days)
Chimerism
(%)
Ultrasensitive RNA
Quantification
(Copies/mL)
Patient Material
ddPCR
(Copies/106Cells)
Gp120V3-Sequence (Dominant
FPRs; Range, %)
HIV-1 LTR HIV-1
Pol
Pre allo-HSCT
−20 15
PBMCs 1967 432 87.2, 89.7 (68.8–96.2)
Tn 1284 167 87.2, 89.7 (87.2–89.7)
Tcm 3074 609 87.2, 89.7 (87.2–91.0)
Ttm 5600 1622 87.2, 89.7 (68.0–89.7)
Tem 6924 1886 87.2, 89.7 (68.8–89.7)
−19 BM 1135 167 87.2, 89.7(73.7–89.7)
Post allo-HSCT
+5 3 PBMCs 949 ND
+16 57 PBMCs 278 <21
+36 100 0 PBMCs <5 ND
+54 95 8 PBMCs 19 ND
+61 PBMCs <20 <20
+65 85
+68
(died) 8 PBMCs <7 <22
Post-mortem biopsies (one biopsy from same site is separated in two parts; 1,2)
+69
Liver, biopsy 1; 2 54; trace ND; <8 No amplification
Lung left, biopsy 1; 2 36; 49 ND; trace 89.7, 87.2 (87.2–89.7)
Lung right, biopsy 1; 2 90; 32 ND; trace 89.7, 87.2 (71.4–92.2)
Spleen, biopsy 1; 2 43; 67 ND; 34 No amplification
Terminal Ileum,
biopsy 1; 2 549; 81 ND; 89 89.7, 87.2 (64.4–89.7)
Allo-HSCT, allogeneic hematopoietic stem cell transplantation. BM, bone marrow. ddPCR, droplet digital PCR.
FPR, false-positive-rate. LTR, long terminal repeat DNA sequence. ND, not done. NTC, no template control.
PBMCs, peripheral blood mononuclear cells. pol, DNA polymerase DNA sequence. Tcm, central memory T-cell.
Tem, effector memory T-cell. Tn, naive T-cell. Ttm, transitional memory T-cell.

Viruses 2022,14, 2069 7 of 12
Viruses 2022, 14, 2069 7 of 13
Twenty days before allo-HSCT, only an unquantifiably low amount of HIV-1 RNA
could be detected with routine diagnostics. Ultra-sensitive plasma viral load was 15
copies/mL (Figure 1a). The viral reservoir was quantified in BM, PBMCs, naïve T-cells,
and all memory T-cell subsets with HIV-1-DNA being most abundant in the more
differentiated T-cell subsets (1135-6924 HIV-1-LTR copies/106 cells) (Table 2).
Figure 1. Clinical and virological data of IciS-05 (a) and IciS-11 (b) pre- and post-allo-HSCT. HIV-1
proviral DNA expressed as LTR DNA copies/million PBMC, and HIV-1 RNA as copies/mL for
plasma and chimerism. Open symbols represent values under the level of quantification.
Abbreviations: allo-HSCT, allogeneic hematopoietic stem cell transplantation. PBMCs, peripheral
blood mononuclear cells.
Table 2. Results of HIV-1-DNA quantification and characterization in patient IciS-05.
Time
Point
(Days)
Chimerism
(%)
Ultrasensitive
RNA
Quantification
(Copies/mL)
Patient
Material
ddPCR
(Copies/106 Cells)
Gp120V3-Sequence
(Dominant FPRs;
Range, %)
HIV-1 LTR HIV-1 pol
Pre allo-HSCT
−20 15
PBMCs 1967 432 87.2, 89.7 (68.8–96.2)
Tn 1284 167 87.2, 89.7 (87.2–89.7)
Tcm 3074 609 87.2, 89.7 (87.2–91.0)
Ttm 5600 1622 87.2, 89.7 (68.0–89.7)
Tem 6924 1886 87.2, 89.7 (68.8–89.7)
−19 BM 1135 167 87.2, 89.7(73.7–89.7)
Post allo-HSCT
+5 3 PBMCs 949 ND
+16 57 PBMCs 278 <21
+36 100 0 PBMCs <5 ND
+54 95 8 PBMCs 19 ND
+61 PBMCs <20 <20
+65 85
+68 (died) 8 PBMCs <7 <22
Post-mortem biopsies (one biopsy from same site is separated in two parts; 1,2)
+69
Liver,
biopsy 1; 2 54; trace ND; <8 No amplification
Lung left,
biopsy 1; 2 36; 49 ND; trace 89.7, 87.2 (87.2–89.7)
Lung right,
biopsy 1; 2 90; 32 ND; trace 89.7, 87.2 (71.4–92.2)
Spleen,
biopsy 1; 2 43; 67 ND; 34 No amplification
Figure 1.
Clinical and virological data of IciS-05 (
a
) and IciS-11 (
b
) pre- and post-allo-HSCT. HIV-1
proviral DNA expressed as LTR DNA copies/million PBMC, and HIV-1 RNA as copies/mL for
plasma and chimerism. Open symbols represent values under the level of quantification. Abbre-
viations: allo-HSCT, allogeneic hematopoietic stem cell transplantation. PBMCs, peripheral blood
mononuclear cells.
In-depth genotypic analyses of the viral reservoir showed that the population was
rather homogeneous with two viral variants dominating all cell types (Supplementary data
Figure S1a). Genotypic analyses predicted CCR5 co-receptor dependency for both variants
(FPR 87.2% and 89.7%) (Table 2). Viruses cultured from PBMCs also showed CCR5-tropism
and were unable to infect MAGI-X4 cells (Supplementary data Figure S1b) and MT2 cells.
After allo-HSCT with CCR5
∆
32 donor cells, a decrease in proviral DNA was observed
in PBMCs (Figure 1a, Table 2). On day +36, when full chimerism was observed in PBLs,
proviral DNA in PBMCs and plasma VL were both below the limit of quantification.
Subsequently, a decrease in chimerism was observed in peripheral T-cells, and HIV-1-DNA
in PBMCs and plasma VL both became detectable on day +54. On day +68, when the
patient deceased, plasma VL was detectable (8 copies/mL) but proviral DNA in PBMCs
was still undetectable. Interestingly, at the same time, proviral DNA could be detected in
all post-mortem biopsies (Table 2). Viral envelope could be amplified and sequenced from
lung tissue and terminal ileum and revealed the dominance of the same CCR5-tropic viral
variants as observed in peripheral blood and BM prior to allo-HSCT (Supplementary data
Figure S1a).
3.2. Patient IciS-11
Shortly before allo-HSCT, low-level plasma HIV-1 RNA was detectable by ultrasen-
sitive viral load assay (2 copies/mL) (Figure 1b). HIV-1-DNA could be detected in BM,
PBMCs, naïve T-cells, and all memory T-cell subsets with most HIV-1-LTR DNA in the
differentiated subsets (73-4629 HIV-1-LTR copies/106 cells) (Table 3).
While proviral DNA levels were lower in comparison to IciS-05, in-depth genotypic
analyses of the viral envelope sequence demonstrated more inter- and intra-sample vari-
ation (Table 3, Supplementary data Figure S1a). All variants were predicted for the use
of the CCR5 co-receptor for viral entry (FPR 9.7–77.3%) (Supplementary data Figure S1a).
HIV-1 variants cultured from BM and PBMCs were also CCR5-tropic and unable to use the
alternative CXCR4 co-receptor as present in MAGI-X4 cells (Supplementary data Figure
S1b) and MT2 cells.
Shortly after allo-HSCT, plasma HIV-1 RNA could no longer be detected and also
PBMC proviral DNA rapidly declined (Table 3, Figure 1b). Over time, with decreasing
chimerism, proviral DNA increased to pre-transplantation levels and plasma ultrasensitive
VL became detectable (3 copies/mL). On day +55 when the graft was rejected, proviral
DNA in PBMCs was twice the pre-transplant level. Considering the relatively low cell
counts after allo-HSCT, a relatively big increase in proviral DNA was observed. In-depth
sequence analyses revealed the same viral sequences as seen before transplantation, and
thus no signs of viral evolution or escape of CCR5 co-receptor usage were observed.

Viruses 2022,14, 2069 8 of 12
Table 3. Results of HIV-1-DNA quantification and characterization in patient IciS-11.
Time
Point
(Days)
Chimerism
(%)
Ultrasensitive RNA
Quantification
(Copies/mL)
Patient Material ddPCR (Copies/106Cells) Gp120V3-Sequence
(Dominant FPR; Range, %)
HIV-1 LTR HIV-1 Pol
Pre allo-HSCT
−22 PBMCs 279 21 18.0 (18.0–70.5)
−20 BM 73 <14 70.5 (39.4–70.5)
−14 2 Tn 571 69 31.8 (18.0–70.5)
Tscm 490 ND 9.7 (9.7–50.3)
Tcm 2222 544 42.4 (22.1–77.3)
Ttm 2780 838 42.4 (18.0–70.5)
Tem 4629 882 21.8 (18.0–39.4)
Post allo-HSCT
+5 0 PBMCs trace <5
+27 60 3 PBMCs 378 ND
+55 0 2 PBMCs 534 72 31.8 (31.8–70.5)
Post 2nd allo-HSCT (days post 1st allo-HSCT)
+11
(+82) 0 PBMCs 8 ND
+27
(+98) 0 PBMCs <2 <4
+35
(+106) 100 0 PBMCs <2 ND
Post-mortem biopsies (one biopsy from same site is separated in two parts; 1,2)
+37
(+108) Liver, biopsy 1; 2 60; <7 ND; trace No amplification
Lung left, biopsy 1; 2 28; <4 ND; <4 No amplification
Lung right, biopsy 1; 2 trace; <3 ND; <3 No amplification
Spleen, biopsy 1; 2 60; <6 ND; <6 No amplification
Brain, biopsy 1; 2 <4; <14 <4; <4 No amplification
38 LN CD4+cells 10 <4 No amplification
Allo-HSCT, allogeneic hematopoietic stem cell transplantation. BM, bone marrow. ddPCR, droplet digital PCR.
FPR, false-positive-rate. LN, lymph node. LTR, long terminal repeat DNA sequence ND, not done. NTC, no
template control. PBMCs, peripheral blood mononuclear cells. pol, DNA polymerase DNA sequence. Tcm,
central memory T-cell. Tem, effector memory T-cell. Tn, naive T-cell. Tscm, stem memory T-cell. Ttm, transitional
memory T-cell.
On day +71, IciS-11 received a second allo-HSCT with heterozygous CCR5
∆
32/WT
donor cells. In the first week after transplantation, a rapid decrease of proviral DNA was
observed. On day +35, when chimerism was 100%, PBMC proviral DNA and
plasma VL
were undetectable. Two days later the patient died of respiratory failure. Multiple post-
mortem biopsies were obtained from liver, lung, spleen, brain, and lymph node. In contrast
to patient IciS-05, detection of HIV-1-DNA in these tissues varied between the different
tissues and biopsies, with lung and brain being negative and other tissues being positive in
only one of the biopsies. The viral envelope could not be successfully amplified from any
of the biopsies (Table 3).
4. Discussion
To date, allo-HSCT is the only strategy to profoundly reduce the HIV-1 reservoir
irrespective of whether CCR5 wild-type stem cells or stem cells lacking expression of the
CCR5 co-receptor for HIV-1 entry are used [2–5,7–9,11,20–22].
Autopsy of patients IciS-05 and IciS-11 provided a unique opportunity to investigate
the impact of allo-HSCT on the dynamics and composition of the viral reservoir in PBMCs
as well as in different tissue compartments. In patient IciS-05 who was transplanted in a
non-myeloablative procedure with CCR5
∆
32/
∆
32 donor cells, no HIV-1-DNA could be
detected by ddPCR in PBMCs but HIV-1-DNA could readily be detected in all biopsies
taken from lung, liver, spleen, and ileum. A study describing post-mortem HIV-1 reservoir

Viruses 2022,14, 2069 9 of 12
analysis in a perinatally infected child who also underwent a myeloablative procedure
using cord blood CCR5
∆
32 donor cells could not detect HIV-1-DNA in blood but could
detect viral DNA in some of the tissues analyzed using sensitive in-situ hybridization
techniques [
21
]. The sensitivity of the ddPCR as employed in the study of Rothenberger
et al. was likely affected by the smaller number of cells that could be assessed from the
tissue biopsies as compared to our study.
In patient IciS-11, who was also transplanted with CCR5
∆
32 donor cells, an initial
decrease of proviral DNA in PBMCs was followed by an increase in proviral DNA perPBMC.
Eberhard et al. showed an HIV-1-specific T-cell activation in IciStem patients after allo-
HSCT and the risk of viral escape from the HIV-1 reservoir shortly after transplantation [
23
].
In-depth sequence analyses of the viral envelope gene revealed identical viral sequences as
seen before transplantation of patient IciS-11 and as such showed no signs of viral evolution
or escape of CCR5 co-receptor usage. As patient IciS-11 experienced cytomegalovirus
(CMV) reactivation after allo-HSCT during incomplete chimerism, we hypothesize that the
increase of proviral DNA could be due to the expansion of particular T-cell populations
after allo-HSCT. This observation is in line with the described expansion of HIV-1-infected
CD4+ T-cells in response to CMV or Epstein–Barr viral antigens, contributing to sustain the
HIV-1-DNA reservoir following chemotherapy [12].
Patient IciS-11 did not suffer from GvHD and, like the Berlin patient, was transplanted
twice. HIV-1-DNA copy number in the tissues was at the detection level of our ddPCR
assays with discrepancies between different tissue biopsies and target regions. These
discrepancies show the difficulties analyzing proviral DNA in high-volume post-mortem
tissues and emphasize the challenges that we face in the accurate detection and quantifica-
tion of proviral DNA in low-volume biopsies of patients undergoing curative strategies.
Though with a successful transplantation full chimerism in the peripheral blood is reached
in the weeks after allo-HSCT, little is known about the turnover and donor chimerism in
tissues. In the Berlin patient, CCR5-positive macrophages were still detected in rectal tissue
more than 5 months after allo-HSCT [
4
]. In patient IciS-11, who died 1 month after his
second transplant, chimerism in PBLs was 100% and no HIV-1-DNA was detected in blood.
Simultaneously, in CD4
+
-cells obtained from the lymph node, where donor chimerism only
reached 38%, HIV-1-DNA was detected, most likely in the recipient cells.
In patient IciS-05, proviral DNA from the different tissue samples was genotypically
characterized and phylogenetic analysis indicated that all tissue-derived sequences in-
termingled with pre-transplantation viral populations as present in the different T-cell
subsets and BM. These data show no evident compartmentalization between viruses ob-
tained from peripheral blood and BM before the transplant compared to viruses obtained
from lung and ileum afterwards. Unlike in the Berlin patient, sequences obtained from
the different post-mortem tissues after allo-HSCT showed no evidence of persistence of
macrophage-tropic viral variants. In general, our data are in agreement with post-mortem
studies demonstrating the lack of compartmentalization of HIV-1 envelope sequences
observed in lung, lymph nodes, and colon [
24
,
25
]. Previous ATI studies performed after
allo-HSCT suggested that the observed viral rebound was caused by pre-existing viral
variants present in the pre-transplant PBLs [
12
,
26
,
27
]. However, these patients’ tissues
were not characterized. Since no HIV-1-DNA-positive cells could be found in blood, the
rebounding viruses post allo-SCT most likely originated from tissue compartments.
A limitation of this study that should be addressed is the short follow-up of the
patients after allo-HSCT. Both patients died shortly after allo-HSCT, hampering the follow-
up of the dynamics of the donor cells in different tissues. Also because sampling occurred
shortly after allo-HSCT, only limited cells were available due to the allo-HSCT procedure.
Analyses of the proviral DNA could only be performed on PBMCs and not on a more
concentrated CD4+ T-cell subset, which could have underestimated the true size of the
peripheral reservoir after allo-HSCT.
In conclusion: This is the first report that performed in-depth post-mortem tissue
quantification and genotypic characterization after allo-HSCT with CCR5
∆
32/
∆
32 donor

Viruses 2022,14, 2069 10 of 12
cells of HIV-1-infected individuals. We demonstrate that the detection of proviral DNA
in tissues was prolonged compared to PBMCs. Tissues clearly play an essential role as
a long-standing viral reservoir and routine sampling in living HIV-1-individuals will be
insufficient to represent the extent of this reservoir. Before conducting ATI, the role of the
tissue reservoir has to be taken into consideration.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/v14092069/s1, Figure S1: Phylogenetic analysis and relative
coreceptor preference.
Author Contributions:
Conceptualization, A.M.J.W., M.N., J.M.-P.; A.M.J.W., and A.S. were involved
in the ethical approval. A.B., P.E., J.H.E.K. and J.T.M.v.d.M. were involved in the clinical process
and provided clinical knowledge. L.A.A.B. performed the autopsy. D.d.J., N.A.N., S.B.H.G., K.T.,
K.B., M.S. and T.M.d.K. generated and analyzed the data. L.E.P.H., A.M.J.W., M.N. and N.A.N. were
involved in draft preparation. L.E.P.H. editing the manuscript. M.S., G.H., M.K., J.D.M., J.T.M.v.d.M.,
A.S.-C., J.J.B., J.S.z.W., J.M.-P., J.H.E.K., A.M.J.W. and M.N. provided scientific guidance and domain
knowledge. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by amfAR, the Foundation for AIDS Research through the
ARCHE program [108930-56-RGRL, 109293-59-RGRL, 109552-61-RGRL], and by the Dutch Aidsfonds
[P-2013034, P-60802, P-13204].
Institutional Review Board Statement:
The study was conducted in accordance with the Decla-
ration of Helsinki, and approved by the Institutional Medical Ethics Committee of the UMCU
(NL35448.041.11 on 27 November 2012 for IciS-05 and NL53114.041.15. on 14 April 2015 for IciS-11).
Informed Consent Statement:
IciS-05 was included after signing a general waiver for the use of
body material for future research, according to the regulations of the UMC Utrecht HSCT Biobank
(HSCT study number 3440). IciS-11 was included after written informed consent was acquired.
Data Availability Statement:
The authors confirm that the data supporting the findings of this study
are available within the article. Further inquiries can be directed to the corresponding author.
Acknowledgments:
The authors thank the following members of the IciStem Consortium who were
not named as authors: Vanderson Rocha (Churchill Hospital, Oxford, United Kingdom); JuliàBlanco
and Jorge Carillo (IrsiCaixa AIDS Research Institute and Institute for Health Science Research Ger-
mans Trias i Pujol (IGTP), Badalona, Spain); Johanna Eberhard and Maximilian Christopeit (University
Medical Center Hamburg-Eppendorf, Hamburg, Germany); Rebeca Bailén and Pascual Balsalobre
(Hospital Gregorio Marañón, Madrid, Spain); Linos Vandekerckhove Marie-Angélique de Scheerder,
and Eva Steel (University of Ghent, Ghent, Belgium); Lisa Barrett, Sharon Oldford, Jill Moore, and
Clarissa Brisseau (NSHA/Dalhousie University, Halifax, Canada); Dieter Häussinger, Guido Kobbe,
and Björn Jensen (University Hospital Düsseldorf, Düsseldorf, Germany); Rolf Kaise and, Elena
Knops (University of Cologne, Cologne, Germany); Marek Widera (University Hospital Essen, Essen,
Germany); Alessandra Bandera, Antonio Muscatello, and Alessandro Soria (San Gerardo Hospital,
Monza, Italy); Gabriella Scarlatti and Simona Piemontese (Istituto Scientifico San Raffaele, Milan,
Italy); Jon Badiola and Manuel Jurado Chacón (Complejo Hospitalario Universitario de Granada,
Granda, Spain); Raquel Saldana (Hospital General de Jerez de la Frontera, Jerez de la Frontera,
Spain); Luz Martín Carbonero (La Paz Hospital, Madrid, Spain); Ildefonso Espigado (University
Hospital Virgen del Rocío, Seville, Spain); Piotr Nowak and Anders Sonnerborg (Karolinska Institutet,
Solna, Sweden); Mitja Nabergoj and Alexandra Calmy (Hôpitaux Universitaires de Genève, Genève,
Switzerland); Kavita Raj, Fabio Cruciani, Varun Mehra, and Carmel Rice (Kings College Hospital,
London, United Kingdom); Angela Bailey (Imperial College Healthcare NHS Trust, London, United
Kingdom); Waseem Qasim (Institute of Child Health & Great Ormond Street Hospital, London,
United Kingdom); Ravindra Gupta (University College London Hospital, London, United Kingdom);
and Koen van Besien (New York Presbyterian Hospital, New York, United States). The authors also
thank Agueda Hernández Rodríguez, from the Microbiology Department of the Hospital Universitari
Germans Trias i Pujol.
Conflicts of Interest:
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts
of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been
disclosed. AJS resports a grant form Dutch Government (ZonMw) and is a committee member for

Viruses 2022,14, 2069 11 of 12
revision Dutch guideline on Sexually Transmitted Diseases in primary care. JJB reports consultancy
fees for the following companies: Avrobio, Omeros, Advanced Clinical, Bluebird Bio, Equillium, Sobi,
and SmartImmune. MS reports grants or contracts from Spanisch Ministry of research and private TV
fundraising funding (MaratóTV3) and payments or honoraria for lectures, presentations, speakers
bureaus, manuscript writing, or educational events from Autonomous University of Barcelona and
GESIDA Spain. ASC report grants or contracts from ANRS, NIH, and MSDAVENIR; payments from
MSD, ViiV healthcare, and Gilead, and a patent for a method for reprogramming CD8+ T-cells to
enhance their therapeutic potential and applications thereof; U.S. Patent application # 63/301, 532
and chair of the Sidaction Scientific and Medical Committee. JHEK reports grants or contracts from
Novartis, Miltenyi, and Gadeta. MN reports grants from Gilead: Rosetta (Global INI resistance);
Dutch Government (ZonMw/NWO): VIMP2, Bridge project; COVIDex-vivo models, Health Holland:
Clear COVID organoids2cureHIV, amfAR: Epigenetic engineering of HIV, NSF: SARS-CoV-2 mutation
frequency and evolution and honoraria for lectures from Virology Education. AW reports grants
from Gilead, Aidfsonds, Dutch Government (ZonMw/NOW), and Health Holland/NLF4 cure, as
well as consulting fees from Gilead, Janssen, Viiv/GSK, and honoraria for lectures from Virology
Education and Southern African HIV Clinicians Society.
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