The document discusses a case study of a 40-year-old man with HIV infection and acute myeloid leukemia who underwent stem cell transplantation using donor cells homozygous for the CCR5 ∆32/∆32 mutation, which provides resistance to CCR5-tropic HIV strains. Post-transplantation, the patient showed reconstitution of CD4+ T cells without detectable HIV RNA or DNA, demonstrating a potential curative approach through this method. The findings emphasize immune system recovery's importance for sustaining the absence of HIV post-treatment and the persistence of CD4+ T cells being susceptible to x4 HIV strains.

1. Cure of HIV infection by CCR5 ∆32/ ∆32
stem cell transplantation
Kristina Allers, Gero Hutter, Jorg Hofmann, Kathrin Rieger,
Christoph Loddenkemper, Eckhard Thiel and Thomas Schneider

2. Human Immunodeficiency Virus (HIV)
• Acquired immunodeficiency
syndrome (AIDS)
• Lentivirus
• HIV-1 and HIV-2

3. Sturcture of HIV
• Single-stranded positive sense RNA
molecule, 9.5 kb in length
• Gag (further cleaved into matrix,
capsid and nucleocapsid)
• Pol (itself cleaved into protease,
reverse transcriptase and integrase)
• Env (160 kDa glycoprotein
cleaved into gp120 and gp41.

4. Pathogenesis
• Loss of activated CD4+ T cells
in the peripheral blood and
lymphoid tissues by
pyroptosis
• Viral entry into CD4 cells is
mediated by the interaction
with a cellular chemokine
receptor, the most common
of which are CCR5 and CXCR4

5. Entry into the cell
• Macrophages and CD4+ T cells
• M-tropic(R5) – CCR5 (50-60%)
• T-tropic(X4) – CXCR4 (<5%)
• Dual tropic (15-20%)
• Mixed tropic (15-20%)

6. CCR5 and gp120 interaction
• gp120 interacts consecutively with CD4
and the CCR5 co-receptor
• Acidic residues and sulfotyrosines in
the amino-terminal domain (Nt) of
CCR5 are crucial for viral fusion and
entry
• Proper post-translational modification
of the CCR5 Nt is required for gp120
binding and viral entry

7. Case Report : 2007
40 year old man
with Acute
Myeloid
Leukemia(AML)
HIV-1 infection -
more than 10
years earlier
HAART for the
previous 4 years
Treatment of the
AML –
Chemotherapy
Severe hepatic
toxicity – HAART
discontinued
viral rebound
(6.9×106 copies
of HIV-1 RNA per
milliliter)
therapy was
resumed and 3
months later,
HIV-1 RNA was
undetectable
7 months later,
acute myeloid
leukemia
relapsed
Stem-cell
transplantation –
homozygous for
CCR5 ∆32/ ∆32
HAART was
administered
until the day
before the
procedure
no active,
replicating HIV
could be
detected 20
months after
HAART had been
discontinued

8. CCR5 ∆32/∆32
32 bp deletion• 32 bp deletion - Chromosome 3
• Homozygous - Naturally resistant to
infection with CCR5- tropic HIV strains
(R5 HIV)
• HIV from the viral reservoir may reseed
the body once the immune system has
efficiently been restored with X4 HIV-
susceptible target cells

9. The curative potential of CCR5 ∆32/ ∆32 SCT for
HIV infection
• The reconstitution of CD4+ T cells at the systemic level as well as in the mucosal
immune system during the post-transplantation period of more than 3.5 years
• To verify the ability of the recovered CD4+ T cells to act as HIV target cells –
• activation status
• CXCR4 expression profile and
• susceptibility to productive HIV infection
• 10 HIV-uninfected SCT patients were included into this study (SCT controls)
• Colon biopsy, liver biopsy, brain biopsy

10. Methods -
Cell preparation and activation –
• Peripheral blood mononuclear
cells (PBMCs) were isolated
from heparinized venous blood
by standard Ficoll gradient
centrifugation.
• Cryoconserved until HIV
susceptibility assays.
Density gradient centrifugation

11. Collagenase digestionCell preparation and activation –
• Mucosal mononuclear cells
(MMCs) were isolated from
colon biopsy specimens by
collagenease type II digestion.
• Cryoconserved until HIV
susceptibility assays.

12. Flow cytometric analysis and cell sorting
• Flow cytometric analysis was
performed by the use of
antibodies against CD3, CD4,
CD31, CD33, CD68, CD38,
CD45RO, CD49d, CD62L, CXCR4
and HLA-DR.
• Lymphocytes were gated on the
basis of characteristic forward
and sideward scatter properties

13. HIV-susceptibility assay
• CCR5-tropic HIV-1 strain JR-CSF and CXCR4-tropic HIV-1 strain
NL4-3 were propagated in PBMC
• Virus-containing cell culture-supernatants were passed
through a 22-µm pore-size filter to remove cell debris and then
treated with Dnase in the presence of 1mM MgCl2 for 30
minutes at 37°C to remove contaminating DNA
• Virus stocks were stored at 80°C

14. HIV - Susceptibility assay
• PBMCs or MMCs were activated with PHA and IL-2 for 48
hours
• The infectious titer of thawed viral stocks was determined by
tissue culture infectious dose 50% assays in PBMC
• Cells were washed and cultivated with virus at a multiplicity of
infection of 0.001 in PMI1640 medium supplemented with 20
U/mL of IL-2
• Analysis of viral replication by quantitative measurement of
the HIV-1 core protein p24 production with the HIV-1 p24
ELISA assay

15. CCR5 genotyping
• Genomic DNA was extracted from sorted mucosal CD4+ T cells
or macrophages with the use of the NucleoSpin TissueXS
• DNA was subjected to PCR amplification with primers for the
CCR5 gene spanning the ∆32-region from nucleotide 826 to
1138 on the chromosome 3p21.31
• The expected fragments were 312 bp for the CCR5 wild-type
and 280 bp for the CCR5 ∆32 variant

16. Results –
Efficient recovery of CD4+ T cells
CD4+ T-cell numbers
• EM - Effector memory
cells
• CM - Central memory
cells
• RTE - Recent thymic
emigrants
• CN - Central naive cells

17. Enrichment of activated/effector memory
CD4+ T cells
• Donor-derived peripheral
CD4+ T cells increased
continuously and, after 2
years, reached levels within
the normal range of age-
matched healthy patients
• Enrichment of activated/
effector memory CD4+ T
cells

18. • CD4+ T-cell expression of
the activation markers CD38,
HLA-DR, and CD49d
• Proliferation marker Ki67 at
9.5 and 24 months after SCT
in comparison with SCT
control and healthy control
patients.
• CD4+ T cells recovered
primarily through
homeostatic proliferation of
memory CD4+ T cells

19. Repopulation of gut mucosal immune system
Immunohistochemical quantification of CD4 T cells in
colon tissue of the CCR5 ∆32/ ∆32 SCT patient, SCT control
patients (27 ± 9 months after transplantation), and
healthy control patients
Genomic DNA was extracted from mucosal
CD4 T cells and subjected to CCR5-specific
PCR spanning the ∆32 region

20. CXCR4 surface availability is not impaired
on recovered CD4+ T cells
• The frequency of CXCR4-expressing cells
within memory CD4 T cells as well as CXCR4
surface expression density at the single cell
level (expressed as the MFI ratio) were
comparable with those of CCR5 wild-type
control patients (80.8% and 6.6%)

21. CXCR4 surface availability is not impaired
on recovered CD4+ T cells
• CXCR4 expression on PHA/IL-2–activated
PBMCs
• Found efficient expression of CXCR4 on
CCR5 ∆32/∆32 CD4+ T cells

22. Recovered CD4+ T cells are susceptible to
productive X4 HIV infection
• Both PBMC’s and MMC’s were
susceptible to productive infection by
X4 HIV
• Virus production of the PBMC-
propagated X4 HIV strain was greater in
peripheral than in mucosal CD4+ T
cells.
• Both peripheral and mucosal CD4+ T
cells were resistant to R5 HIV infection

23. • 12 months after CCR5 ∆32/
∆32 SCT, CCR5-expressing
CD4 T cells or
macrophages/Kupffer cells
were not detectable
Long-lived HIV target cells of host origin
were replaced with donor-derived cells

24. • 17 months after CCR5 ∆32/ ∆32
SCT, no CCR5-expressing
macrophages/microglia were
found in the brain
Long-lived HIV target cells of host origin
were replaced with donor-derived cells

25. • Presence of CCR5-expressing
macrophages at 5.5 months
after CCR5 ∆32/ ∆32 SCT
• Noncirculating immune cells
• Virtually chemo/ radio-resistant
• Possible viral reservoirs
Long-lived HIV target cells of host origin
were replaced with donor-derived cells

26. • The presence of HIV RNA and
HIV DNA was examined in
distinct tissue compartments
during the course of 45 months
after CCR5 ∆32/ ∆32 SCT.
• Viral sequences were not
detectable in all the samples
tested
HIV remains undetectable in distinct tissue
compartments

27. • Immune reconstitution is critical to the long-term success of the SCT
• In the CCR5 ∆32/ ∆32 SCT patient, CD4 T-cell numbers have even returned to
the normal range of healthy patients whereas HIV RNA and HIV DNA remain
continuously undetectable in plasma and PBMC.
• Although the recovered CD4+ T cells are susceptible to infection with X4 HIV,
the patient remains without any evidence of HIV infection for more than 3.5
years after discontinuation of ART.
Discussion –