Is it true? Stem cells' mutation cures AIDS - (op ed with fulltext links)

That would be CCR5-delta32: that's the same rare mutation for which the historical record shows an increase in frequency, suddenly, from around one in 40,000 to one in five. That was no typo: CCR5-delta32 went from one quarter of a thousandth of a per cent to 20 per cent. When? Why the middle ages of course. Where? Northern Europe. Why? Natural selection. Everyone without the mutation died.

From the Black Death.

Happens to protect select caucasian homo sapiens from the very AIDS killing so many of those pesky brown people whose unchecked fecundity has been long feared by the power elite as the biggest threat to the balance of power on this planet. Because it surely is -- if bombs or tech or government repression or aliens or anarchy or famine or diseases don't take us out first. Kind of makes you a little paranoid, don't it? Never mind that Doctor, what was his name?, who boasted to the US government that he could engineer a virus that used the human immune system against itself -- and selected target population by phenotype -- in five to ten years -- in 1971 I think it was. Dismiss as apocryphal stories about the two clinics that surviving LGBT old-timers remember, clinics that sprung up out of nowhere, one in SF and one in NYC, with an experimental cure for Hepatitis C and a request for volunteers to test it. Clinics which disappeared equally suddenly, and whose patients all died shortly thereafter, the first victims of AIDS, a disease that the mainstream medical establishment could not even successfully categorize, much less treat, until many years and deaths later. A disease with social taboo on its side, soon to make itself at home in Africa. You can't ignore this detail, I'd wager.

The mutation that confers immunity to the plague just so happens to protect against AIDS.

What a coincidence! Which reminds me (and this ought to really cook your noodle):

It protects against H1N1 as well.

Have a nice day.

Evidence for

the cure of

HIV infection by CCR5Δ32/Δ32

stem cell transplantation

doi:10.1182/blood-2010-09-309591 Prepublished online Dec 8, 2010;

Thomas Schneider

Kristina Allers,

Gero Hütter,

Jörg Hofmann,

Christoph Loddenkemper,

Kathrin Rieger,

Eckhard Thiel

and

Thomas Schneider

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. Hematology; all rights reservedCopyright 2010 by The American Society of 20036.

the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

. Hematology; all rights reservedCopyright 2010 by The American Society of 20036.

the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

1

: Department of Gastroenterology, Infectious Diseases, and Rheumatology, Medical Clinic I,

Campus Benjamin Franklin, Charité - University Medicine Berlin, Germany

2

: Department of Hematology, Oncology, and Transfusion Medicine, Medical Clinic III,

Campus Benjamin Franklin, Charité - University Medicine Berlin, Germany

3

: Institute of Medical Virology, Helmut-Ruska-Haus, Campus Mitte, Charité - University

Medicine Berlin, Germany

4

: Institute of Pathology/Research Center ImmunoSciences (RCIS), Campus Benjamin

Franklin, Charité - University Medicine Berlin, Germany

5

: Current address: Institute of Transfusion Medicine and Immunology, University

Heidelberg, Germany

6

: Current address: Institute of Pathology, Technische Universität München, Munich,

Germany

Running title: CURE OF HIV INFECTION BY CCR5Δ32/Δ32 SCT

Corresponding author: Kristina Allers

Medical Clinic I - Gastroenterology, Infectious Diseases and

Rheumatology

Charité - Campus Benjamin Franklin

Hindenburgdamm 30

12203 Berlin

Germany

Phone: +49 30 8445 2743, Fax: +49 30 8445 2903

Email: kristina.allers@charite.de

Blood First Edition Paper, prepublished online December 8, 2010; DOI 10.1182/blood-2010-09-309591

Copyright © 2010 American Society of Hematology

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ABSTRACT

HIV entry into CD4+ cells requires interaction with a cellular receptor, generally either

CCR5 or CXCR4. We have previously reported the case of an HIV-infected patient in

whom viral replication remained absent despite discontinuation of antiretroviral

therapy after transplantation with CCR5Δ32/Δ32 stem cells. However, it was

expected that the long-lived viral reservoir would lead to HIV rebound and disease

progression during the process of immune reconstitution. In the present study, we

demonstrate successful reconstitution of CD4+ T cells at the systemic level as well as

in the gut mucosal immune system following CCR5Δ32/Δ32 stem cell transplantation,

while the patient remains without any sign of HIV infection. This was observed

although recovered CD4+ T cells contain a high proportion of activated memory CD4+

T cells, i.e. the preferential targets of HIV, and are susceptible to productive infection

with CXCR4-tropic HIV. Furthermore, during the process of immune reconstitution,

we found evidence for the replacement of long-lived host tissue cells with donor-

derived cells indicating that the size of the viral reservoir has been reduced over time.

In conclusion, our results strongly suggest that cure of HIV has been achieved in this

patient.

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Introduction

Destruction of the immune system by the human immunodeficiency virus (HIV) is driven by

the loss of CD4+ T cells in the peripheral blood and lymphoid tissues. Viral entry into CD4+

cells is mediated by the interaction with a cellular chemokine receptor, the most common are

CCR5 and CXCR4.1 Since subsequent viral replication requires cellular gene expression

processes, activated CD4+ cells are the primary targets of productive HIV infection.

Consequently, HIV infection leads predominantly to the depletion of activated memory CD4+

T cells, the vast majority of which reside in the gastrointestinal (GI) mucosa.2-4 Although

therapeutical control of HIV replication allows the immune system to partially restore and

delays disease progression, cure of HIV infection remains still unachievable with the

currently available antiretroviral drugs. The major barrier to viral eradication in patients

receiving antiretroviral therapy (ART) is the establishment of HIV reservoirs including low-

level productively and latently infected cells.5-7 Thus, maintenance of replication-competent

HIV in long-lived cells and distinct anatomical sanctuaries allows the virus to reseed the body

once ART is discontinued.8

Cells of individuals homozygous for the CCR5 gene variant Δ32 (CCR5Δ32/Δ32) are

naturally resistant to infection with CCR5-tropic HIV strains (R5 HIV) due to the lack of

CCR5 cell surface expression.9 Previously, we demonstrated the feasibility of hematopoietic

stem cell transplantation with CCR5Δ32/Δ32 donor cells (CCR5Δ32/Δ32 SCT) in an HIV-

infected patient with relapsed acute myeloid leukemia (AML) and documented absent viremia

during the first 20 months of remission while the patient did not receive ART.10,11 This case

clearly emphasizes the importance for continuing research in the field of CCR5 targeted

treatment strategies, but uncertainty remained over whether cure of HIV infection has been

achieved in this patient.

In the setting of HIV infection, the effects of pre-transplant conditioning do not allow the

complete elimination of HIV, as demonstrated by previous studies showing that HIV-infected

patients with a stem cell transplant generally experience viral rebound when ART is

discontinued.12-17 For this reason, together with the fact that CXCR4-tropic HIV variants (X4

HIV) were present within the patient’s pre-transplant HIV population, it was reasonable to

hypothesize that HIV from the viral reservoir may reseed the body once the immune system

has efficiently been restored with X4 HIV susceptible target cells.18,19

Accordingly, key questions that remain to be answered are (i) whether CD4+ T cells have

been efficiently restored throughout the body (ii) whether or not the patient’s immune system

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includes HIV susceptible target cells, and (iii) how stable the size of the HIV reservoir is

during the process of immune reconstitution following CCR5Δ32/Δ32 SCT.

Here, to address these questions, we extend our previous study in order to improve our

knowledge about the curative potential of CCR5Δ32/Δ32 SCT for HIV infection. We

evaluated the reconstitution of CD4+ T cells at the systemic level as well as in the mucosal

immune system during the posstransplant period of more than 3.5 years. In order to verify the

ability of the recovered CD4+ T cells to act as HIV target cells, their activation status, CXCR4

expression profile and susceptibility to productive HIV infection was analyzed. Moreover, as

the absence of the CCR5 wild-type gene variant in donor cells gave the possibility to

discriminate between donor- and host-derived immune cells, we were able to examine the

persistence of potential viral reservoirs, in addition to the detection of viral sequences, in

distinct tissue compartments.

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Subjects, Materials, and Methods

Subjects

In February 2007, an HIV-infected patient underwent stem cell transplantation (SCT) due to a

relapse of AML with a graft consisting of CCR5Δ32/Δ32 donor cells. The pre-transplant

conditioning regimen included 100 mg/m2 of amsacrine, 30 mg/m2 of fludarabine, 2 g/m2 of

cytarabine (day -12 until -9), 60 mg/kg of cyclophosphamide (days -4 and -3), 5.5 mg/kg of

rabbit antithymocyte globuline (in three doses between day -3 and -1), and a 400 cGy total

body irradiation (TBI; day -5). ART was discontinued on the day of transplantation, and 13

months later the patient received a second transplant with CCR5Δ32/Δ32 stem cells from the

same donor due to a second relapse of AML. The conditioning regimen consisted of 100

mg/m2 of cytarabine (day -7 until day -1), 6 mg/m2 of gemtuzumab (day -7 and day -1), and a

200 cGy TBI (day -1). For clinical data and further details, see Hütter et al..10 At 5.5, 24, and

29 month following the first CCR5Δ32/Δ32 SCT, the patient underwent colonoscopy and

biopsy specimens were taken due to suspected intestinal graft-versus-host disease (GvHD)

while tapering immunosuppressive treatment. With the patient’s informed consent for this

procedure, 10 to 13 additional colon biopsy specimens were collected at each time point for

research purpose of the present study. Examination of histological colon sections excluded the

diagnosis of intestinal GvHD. Twelve months post-transplant, the patient underwent liver

biopsy and histological examination confirmed GvHD grade I that was controlled with

adaption of immunosuppressive therapy (cyclosporine A, methylprednisolone, mycophenolate

mofetil). 17 months post-transplant, the patient presented with neurological disorders.

Magnetic resonance imaging of the brain identified signal abnormalities compatible with

leukoencephalopathy. For further evaluation, cerebrospinal fluid (CSF) samples were

collected repeatedly and a brain biopsy was performed, additionally. PCR detection of JC

virus was negative in all samples. Histological evaluation revealed astrogliosis with

microglial activation. The cumulative effect of initial AML treatment chemotherapy and

salvage chemotherapy after relapse of AML, as well as pre-transplant conditioning regimen

including TBI were assumed as cause of leukoencephalopathy,20 which turned out to be self-

limiting. Immunosuppressive treatment has been stopped 38 months after CCR5Δ32/Δ32 SCT

without recurrence of GvHD.

In addititon, 10 HIV-uninfected SCT patients were included into this study (SCT controls).

Four of these patients underwent colonoscopy, and intestinal GvHD was histologically

excluded in all cases. 15 HIV-uninfected individuals served as healthy controls, five of them

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underwent colonoscopy for cancer preventive examination. The study was approved by the

Charité - University Medicine Berlin institutional review board, and all participants gave

informed consent to study participation in accordance with the Declaration of Helsinki.

Cell preparation and activation

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by

standard Ficoll gradient centrifugation, and mucosal mononuclear cells (MMC) were isolated

from colon biopsy specimens by collagenease type II (Sigma) digestion.21 Cells were either

immediately used for subsequent analysis or cryoconserved until HIV susceptibility assays.

For some experiments, PBMC were activated for two days with 3 μg/ml of

phythemagglutinin (PHA; Sigma) and 50 U/ml of recombinant interleukin-2 (IL-2; R&D

Systems) in RPMI 1640 + GlutaMAX cell culture medium (Invitrogen) containing 10% heat-

inactivated fetal calf serum (Sigma), 100 U/ml of penicillin, and 100 μg/ml streptomycin

(both from Biochrom) before flow cytometric analysis.

Flow cytometric analysis and cell sorting

Flow cytometric analysis was performed by using antibodies against CD3 (clone UCHT1; BD

Biosciences), CD4 (SK3; BD), CD31 (WM59; BD), CD38 (HIT2; BD), CD45RO (UCHL1;

BD), CD49d (9F10; BD), CD62L (Dreg-56; BD), CXCR4 (12G5; BD), HLA-DR (Immu357;

Beckman Coulter), and Ki67 (Ki67; DAKO). Absolute numbers of CD4+ T cells were

determined in fresh whole blood by the use of TruCount tubes and CD3/CD4/CD8 TriTest

(BD) according to the manufacturer’s protocol. Data were acquired on the FACSCalibur flow

cytometer (BD) and analyzed with CellQuest software (BD). Lymphocytes were gated on the

basis of characteristic forward and sideward scatter properties. Central memory CD4+ T cells

(CM) were classified by co-expression of CD45RO and CD62L and effector memory CD4+ T

cells (EM) were classified by lack of CD62L. Recent thymic emigrants (RTE) were identified

by co-expression of CD31 and CD62L on CD45RO- CD4+ T cells and central naïve CD4+ T

(CN) cells by lack of CD31.22 CXCR4 expression density on CD4+ T cells was evaluated as

the mean fluorescence intensity (MFI) of CXCR4 expression divided by the MFI value

obtained with the corresponding isotype control (BD) and is expressed as the MFI ratio.

For mucosal cell sorting, the following additional antibodies were used: anti-CD33

(AC104.3E3; Miltenyi Biotec) and anti-CD68 (Kim7; BD). Cell sorting procedures were

performed by customer service of the Flow Cytometry Core Facility at the Berlin-

Brandenburg Center for Regenerative Therapies, Germany, with the use of the FACSAriaII

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flow cytometer (BD) and FACSDiva software (BD). Mucosal CD4+ T cells were identified by

their co-expression of CD3 and CD4 in the lymphocyte gate and mucosal macrophages were

selected by their co-expression of CD33 and CD68 in the CD3- macrophage gate.23

Antibodies were conjugated to fluorescin, phycoerythrin, peridinin chlorophyll protein, or

allophycocyanin.

HIV susceptibility assay

CCR5-tropic HIV-1 strain JR-CSF (obtained from the EVA Centre for AIDS Reagents,

NIBSC, UK) was propagated in PBMC. A stock of CXCR4-tropic HIV-1 strain NL4-3 was

generated from the HIV-1 molecular clone pNL4-3 (obtained from the EVA Centre for AIDS

Reagents), and then propagated in PBMC. Virus-containing cell culture supernatants were

passed through a 22 μm-pore-size filter (BD) to remove cell debris and then treated with

Dnase (Boehringer Mannheim) in the presence of 1 mM MgCl2 for 30 min at 37 °C to remove

contaminating DNA. Virus stocks were stored at –80 °C. The infectious titer of thawed viral

stocks was determined by tissue culture infectious dose 50% assays in PBMC. Prior to

infection, PBMC or MMC were activated with PHA and IL-2 for 48 h. Cells were washed and

cultivated with virus at a multiplicity of infection (MOI) of 0.001 in RPMI1640 medium

supplemented with 20U/ml of IL-2. Viral stocks diluted in cell free medium served as

background control, the patient’s cells alone as mock control, and cell-free virus suspensions

as control for background corrections. Supernatants were removed from cell cultures and cell-

free controls as indicated, being replaced by fresh medium, and stored at –80 °C till analysis

for viral replication by quantitative measurement of the HIV-1 core protein p24 production by

using the HIV-1 p24 ELISA assay (XpressBiotech) according to the manufacturer’s protocol.

Immunohistochemistry and immunofluorescence staining

Immunostaining on paraffin sections was performed as described before.24 Primary antibodies

were mouse anti-CD4 (1F6; Novocastra), mouse anti-CD68 (PGM1; DAKO) or goat anti-

CCR5 (CKR-5 (C20); Santa Cruz Biotechnology). For detection of CD4 labeling the

Streptavidine Alkaline Phosphatase-kit (DAKO) was used. Positive cells within the mucosa of

colon tissue were quantified per high power field (hpf, 0.237 mm2), and 10 hpf were averaged

in each case. Per sampling at least three sections were analyzed. Immunohistochemical

evaluations were performed in a blinded manner, i.e. unaware of the patient’s clinical

characteristics. For CD4/CCR5 or CD68/CCR5 double immunofluorescence labeling, Alexa-

Fluor 488-conjugated anti-mouse was used in combination with Alexa-Fluor 555-conjugated

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anti-goat (Invitrogen). Images were acquired using a fluorescence microscope (AxioImager

Z1) equipped with a charged-coupled-device camera (AxioCam MRm) and processed with

Axiovision software (Carl Zeiss MicroImaging, Inc.). Negative controls were performed by

omitting the primary antibodies, and unspecific staining of the antibodies was excluded by

using isotype control antibodies.

CCR5 genotyping

In order to study the CCR5 gene variant in HIV target cells, genomic DNA was extracted

from sorted mucosal CD4+ T cells or macrophages with the use of the NucleoSpin TissueXS

(Macherey & Nagel) according to the manufacturer’s protocol. DNA was then subjected to

PCR amplifcation employing primers for the CCR5 gene spanning the Δ32-region from

nucleotide 826 to 1138 on the chromosome 3p21.31 (acc.-nr.: NM_000579). The expected

fragments were 312 bp for the CCR5 wild-type and 280 bp for the CCR5Δ32 variant.

Detection of HIV and HIV specific antibodies

Viral RNA was isolated from plasma or CSF and the long terminal repeat (LTR) and gag

regions were amplified and detected with the use of the COBAS® AmpliPrep/COBAS®

TaqMan HIV-1 Test v1.0 (Roche). Total DNA was isolated from PBMC, tissue biopsy

specimens and sorted cells with the use of the QIAamp DNA Blood Mini Kit, the Allprep

DNA/RNA Mini Kit (both from Qiagen) and the NucleoSpin Tissue XS, respectively,

following the manufacturer’s directions and the LTR and env regions were detected as

described previously.10 Antibodies directed against HIV antigens in serum samples were

detected with immunoblot (Abbott) as described before.10

Statistical analysis

Data are represented as medians and were analyzed wit the use of two-tailed Student t test

with Prism software version 4.0 (Graph Pad Inc.). Significance is denoted with asterisks (i.e.

*P <>P <>P <>

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Results

Efficient recovery of CD4+ T cells was associated with a characteristic

enrichment of activated/effector memory CD4+ T cells

Following CCR5Δ32/Δ32 SCT, chimerism analysis as well as genotyping of CCR5 alleles

suggested that host T cells were completely eliminated from the periphery.10 Numbers of

donor-derived peripheral CD4+ T cells increased continuously and, after two years, reached

levels within the normal range of age-matched healthy individuals (Figure 1A). Further

phenotypic analysis revealed an increase of memory CD4+ T cell numbers, with a parallel, but

low, increase of CD4+ recent thymic emigrant as well as central naïve CD4+ T cell numbers.

In both the CCR5Δ32/Δ32 SCT patient and the SCT control patients, the proportion of central

memory CD4+ T cells was within the normal range, whereas effector memory CD4+ T cells

remained markedly enriched within the CD4+ T cell compartment as compared with healthy

control values (Figure 1A, B). This cellular composition indicates a proliferative expansion of

mature CD4+ T cells. In accordance, the frequency of cells expressing the activation markers

CD38, CD49d and HLA-DR and the proliferation marker Ki67 was higher within CD4+ T

cells from CCR5Δ32/Δ32 SCT and control SCT patients than from healthy controls (Figure

1C). Thus in both cases, CD4+ T cells recovered primarily through homeostatic proliferation

of memory CD4+ T cells, confirming previous reports of post-transplant immune

reconstitution.25,26 These results demonstrate that the CCR5Δ32/Δ32 SCT patient experienced

a regular reconstitution of the peripheral CD4+ T cell compartment following CCR5Δ32/Δ32

SCT, including the characteristic enrichment of activated/effector memory CD4+ T cells.

Donor-derived CD4+ T cells have efficiently repopulated the gut mucosal

immune system

Most of the body’s CD4+ T cells reside in the GI tract. In order to assess the recovery of CD4+

T cells in the gut mucosal immune system, CD4+ T cells were immunohistochemically

quantified in colon tissue sections at three time points after CCR5Δ32/Δ32 SCT and were

compared with SCT controls and healthy controls. The number of mucosal CD4+ T cells

increased during the post-transplant period and 29 months after CCR5Δ32/Δ32 SCT, the

density of CD4+ T cells in the GI mucosa was similar to that of the SCT control patients (162

vs. 180 ± 33 cells/hpf; Figure 2A). Thus, no lack of immune reconstitution could be noted in

the mucosal immune system. Interestingly, compared to healthy controls there was a more

than twofold-increased frequency of mucosal CD4+ T cells in all SCT patients (60 ± 12 vs.

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162 ± 29 cells/hpf) demonstrating that treatment with conditioning followed by SCT triggers

the enrichment of HIV target cells in the gut mucosal immune system (Figure 2A).

In order to confirm the donor-origin of mucosal CD4+ T cells, we performed additional

phenotypic and genotypic analysis. In situ detection of CCR5 by immunofluorescence

staining at 5.5 and 24 months after CCR5Δ32/Δ32 SCT revealed no CCR5 expression on

mucosal CD4+ T cells (not shown), which corroborates our previous finding from flow

cytometric analysis10. Moreover, CD4+ T cells sorted from MMC at 24 and 29 months after

CCR5Δ32/Δ32 SCT were negative for the CCR5 wild-type gene (Figure 2B). This

demonstrates that increased numbers of mucosal CD4+ T cells were exclusively derived from

donor hematopoietic cells. Taken together, these results reveal that circulating donor-derived

CD4+ T cells were efficiently recruited to the gastrointestinal tract and have repopulated the

mucosal CD4+ T cell compartment following CCR5Δ32/Δ32 SCT.

CXCR4 surface availability is not impaired on recovered CD4+ T cells

Reconstitution of the CD4+ T cell compartment following CCR5Δ32/Δ32 SCT was associated

with an expansion of activated memory cells (Figures 1 and 2), the preferential targets of

productive HIV infection. Donor-derived CD4+ T cells are naturally resistant to CCR5-tropic

HIV infection due to the lack of CCR5 surface expression. We were interested in whether

recovered CCR5Δ32/Δ32 CD4+ T cells might additionally exhibit reduced CXCR4 surface

availability. Therefore, we analyzed fresh whole blood cells and MMC for CXCR4 surface

expression on CD4+ T cells in comparison with cells obtained from CCR5 wild-type

individuals. As shown in figure 3A, both 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 ration) were comparable to those of CCR5 wild-type controls (80.8 ±

2.0% and 6.6 ± 1.0, respectively). This was also observed for the peripheral naïve CD4+ T cell

compartment (not shown). Since the level of CXCR4 expression may vary with cell

activation, we next analyzed CXCR4 expression on CD4+ T cells upon ex vivo activation and

found efficient expression of CXCR4 on CCR5Δ32/Δ32 CD4+ T cells (Figure 3B). These data

demonstrate that the CCR5Δ32/Δ32 SCT was not associated with an impaired CXCR4

expression on recovered CD4+ T cells. In vivo, the availability of CXCR4 may be affected by

the chemokine CXCL12, the physiologic ligand of CXCR4.27 During the immune

reconstitution period, CXCL12 plasma levels in the CCR5Δ32/Δ32 SCT patient remained

within the normal range of healthy individuals (not shown) indicating that the in vivo

availability of CXCR4 was not impaired by naturally occurring receptor occupation.

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Altogether, these results indicate that recovered CD4+ T cells are not protected against X4

HIV entry.

Recovered CD4+ T cells are susceptible to productive X4 HIV infection

Susceptibility of recovered CD4+ T cells in the central as well as the mucosal immune system

to productive HIV infection was studied by ex vivo infections of PBMC and MMC obtained

after CCR5Δ32/Δ32 SCT. As shown in figure 4, cells from both compartments were

susceptible to productive infection by X4 HIV. Consistent with our previous observation,

virus production of the PBMC-propagated X4 HIV strain was higher in peripheral than in

mucosal CD4+ T cells.28 As expected, due to the lack of CCR5 surface expression on donor-

derived cells, both peripheral and mucosal CD4+ T cells were resistant to R5 HIV infection.

Long-lived HIV target cells of host-origin were replaced with donor-derived

cells during the post-transplant period

Owing to the fact that recovered CD4+ T are susceptible to productive X4 HIV infection,

long-lived HIV-infected host cells that survived the chemo- and irradiation therapies represent

potential sources for HIV emerge. Non-circulating immune cells such as tissue CD4+ T cells

or macrophages are virtually chemo/radio-resistant and, therefore, represent possible viral

reservoirs. We investigated the presence of residual host immune cells after CCR5Δ32/Δ32

SCT by in situ immunofluorescence detection of cellular CCR5 expression. Clinical samples

from the liver, the brain and the colon could be used for research purpose of the present study

after diagnosis has been done. Brain tissue specimens were available from the white matter

and the cortex. From the colon, three separate biopsy specimens were available from each of

three time points during the course of immune reconstitution. In the liver, 12 months after

CCR5Δ32/Δ32 SCT, CCR5-expressing CD4+ T cells or macrophages/Kupffer cells were not

detectable (Figure 5A). Likewise, 17 months after CCR5Δ32/Δ32 SCT, no CCR5-expressing

macrophages/microglia were found in the brain (Figure 5B).

In the colon, there was no evidence of residual host CD4+ T cells after CCR5Δ32/Δ32 SCT, as

already described above (Figure 2B). However, in situ immunofluorescence staining revealed

the presence of CCR5-expressing macrophages at 5.5 months post CCR5Δ32/Δ32 SCT,

which is in agreement with our previous flow cytometric data10 and demonstrates the

persistence of host macrophages during the first months after CCR5Δ32/Δ32 SCT (Figure

6A). Importantly, later in the course of immune reconstitution, CCR5 expression on

macrophages became undetectable indicating their replacement with donor-derived cells

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(Figure 6A). To further prove the origin of mucosal macrophages, we performed additional

genotypic analysis of sorted mucosal macrophages. As shown in figure 6B, 24 and 29 months

after CCR5Δ32/Δ32 SCT, mucosal macrophages were negative for the CCR5 wild-type gene.

The absence of host’s genomic DNA in mucosal macrophages at these time points confirms

the phenotypic results and suggests that host macrophages have been replaced with donor-

derived cells during the posttranplant period.

HIV remains undetectable in distinct tissue compartments

The presence of HIV RNA and HIV DNA was examined in distinct tissue compartments over

45 months following CCR5Δ32/Δ32 SCT. Viral sequences were not detectable in all the

samples tested (Table 1).

Antibodies against HIV decrease over time

Previously, we reported the loss of antibodies directed against the HIV polymerase as well as

a decline of HIV envelope and core specific antibodies during the first 20 months after

CCR5Δ32/Δ32 SCT.10 Immunoblot analysis revealed a continuing decline of HIV specific

antibodies thereafter demonstrating the process of serodeconversion: whereas HIV core-

directed antibodies (p17, p24) have disappeared completely, the serum level of antibodies

against the HIV envelope (gp41, gp120) has further decreased. Today, the patient has only

HIV envelope specific antibodies.

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Discussion

Immune reconstitution is critical to the long-term success of the stem cell transplant and, in

HIV-infected patients, additionally provides a prerequisite for viral rebound and HIV disease

progression. Progressive infection in turn impairs the reconstitution of CD4+ T cells after

SCT. Our results show, that systemic recovery of CD4+ T cells following CCR5Δ32/Δ32 SCT

and discontinuation of ART was not impaired when compared to that of SCT control patients.

In accordance with previous studies,25,26 repopulation of the CD4+ T cell compartment was

associated with peripheral expansion of donor-derived memory CD4+ T cells, that probably

occurs in order to compensate for the limited thymic capacity in adults.29-31 Generally, this

homeostasis-driven expansion of activated memory CD4+ T cells leads to an enrichment of

the preferential targets for productive infection with both R5 HIV and X4 HIV32 and likely

contributes to the rapid dynamic of HIV rebound following conventional SCT in HIV-

infected patients.12,14,15,17 Viral tropism analysis was not in the focus of previous reports of

HIV infected patients with conventional SCT and would be an interesting issue to address in

future studies.

In the CCR5Δ32/Δ32 SCT patient, CD4+ T cell numbers have even returned to the normal

range of healthy individuals whereas HIV RNA and HIV DNA remain continuously

undetectable in plasma and PBMC, respectively. Today, by monitoring the most common

prognostic markers, i.e. plasma viral load and CD4+ T cell counts in the peripheral blood, HIV

disease cannot be assessed in this patient.

However, observations from the central immune compartment need not be representative for

distinct tissue compartments throughout the body. Only 1-2% of the body’s total CD4+ T cells

reside in the peripheral blood whereas the vast majority of immune cells are located in the GI

tract.33 Containing most of the body’s activated memory CD4+ T cells with high expression of

cellular receptors, the mucosal immune system is highly prone to productive infection with

both R5 HIV and X4 HIV.3,28,34-36 In fact, profound depletion of CD4+ T cells in the GI

mucosa occurs earlier than that in blood or lymph nodes regardless of the infection route, and

even with complete suppression of viremia for many years, residual low-level replication in

the GI tract prevents full recovery of mucosal CD4+ T cells in ART-treated HIV-infected

individuals.2,37-39 Poor recovery of CD4+ T cells in the mucosal immune system is therefore an

important risk factor for the development of HIV disease progression. Following

CCR5Δ32/Δ32 SCT, we found that the process of immune reconstitution included a gradual

increase of donor-derived CD4+ T cells in the GI mucosa. When compared to HIV-uninfected

SCT controls, mucosal CD4+ T cell numbers normalized whereas HIV remains undetectable

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in gut tissue specimens as well as in mucosal HIV target cell populations. These findings

argue for the absence of HIV disease progression in the largest component of the lymphoid

organ system. Surprisingly, compared with healthy controls, mucosal CD4+ T cell numbers in

both the CCR5Δ32/Δ32 SCT patient and the SCT control patients were increased. This may

likely be explained by the high prevalence of activated/effector memory CD4+ T cells in the

circulation, for which we have previously found enhanced gut-homing capacity.40 In addition,

the normalized frequency of central memory cells within circulating CD4+ T cells suggests

that recovered CD4+ T cells have been efficiently directed to peripheral lymph nodes.41,42

Furthermore, the decline of HIV-specific antibodies following CCR5Δ32/Δ32 SCT indicates

the continuous absence of HIV gene expression in lymphoid tissues after discontinuation of

ART.

In addition to their natural protection from R5 HIV infection, CCR5Δ32/Δ32 CD4+ T cells of

some individuals have been suggested to be less susceptible to X4 HIV entry as a result of

down-regulated CXCR4 expression.43,44 However, in the patient described here, we found no

evidence for an abnormal CXCR4 expression on recovered CD4+ T cells. Moreover, the

patient’s peripheral and mucosal CD4+ T cells are susceptible to productive infection with X4

HIV demonstrating that the CCR5Δ32/Δ32 SCT has not provided protection against X4 HIV

infection. Consequently, the patient’s risk of exogenous HIV re-infection is not completely

eliminated.

Altogether, our results demonstrate that the process of immune reconstitution has successfully

restored both the central and the mucosal immune system with CD4+ T cells that lack CCR5

surface expression but have susceptibility to productive X4 HIV infection. Consequently, host

cells that survived the chemo-irradiation therapies represent potential sources for X4 HIV

rebound. Host-originating CD4+ T cells appear to be completely removed from the patient’s

immune system, however, in particular tissue macrophages may play a critical role as viral

reservoir because they are virtually resistant to conditioning procedures and less prone to the

cytopathic effects of HIV infection.45 HIV became not detectable in the brain during a

neuropathological episode although the associated microglia activation and astrogliosis may

support re-activation of viral replication from latently infected cells. This provides indirect

evidence for the absence of replication-competent HIV in cells of the brain. Furthermore, in

brain as well as in liver tissue sections, no CCR5 expression on macrophages was detectable

indicating the replacement of host microglial cells and Kupffer cells by donor-derived cells.

Because CCR5 is not constitutively expressed on tissue macrophages46, the limited sample

availability did not allow us to extend the phenotypic results to cell-specific genomic analysis,

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and also, the analyzed sections are representative only for a very limited area of the respective

organ, these findings cannot definitely exclude the presence of residual, potentially infected,

host cells. However, there is convincing evidence from studies in mice to suggest that host

tissue macrophages were efficiently replaced with donor-derived cells during the course of

immune reconstitution. For example, while it is generally accepted that microglia under

steady-state conditions are very slowly renewed by cells of hematopoietic origin, it has been

demonstrated that the conditioning procedure efficiently enhances this process after stem cell

transplantation.47,48 Moreover, the majority of Kupffer cells are replaced already early after

SCT49 and, importantly, increasing conversion rates of tissue macrophages over time

following transplantation has been demonstrated in distinct tissue compartments throughout

the whole body.50,51 Evidence in support of the conclusion that conversion from host to donor

tissue macrophages took place in the patient following CCR5Δ32/Δ32 SCT comes from our

serial analysis in colon tissue. Here, phenotypic results revealed that residual host cells were

present within the mucosal macrophage population during the first months after

CCR5Δ32/Δ32 SCT. Later in the course of immune reconstitution, host-originating

macrophages became undetectable in the GI mucosa by both phenotypic and genotypic

analysis. These findings suggest that the replacement of host tissue cells with donor-derived

cells has reduced the size of the viral reservoir during the course of immune reconstitution

and, consequently, has lowered the risk of HIV rebound over time. Cell replacement in tissues

under post-transplant conditions may even allow for complete eradication of HIV, however,

the unfeasibility to analyze every single cell in living humans rules out the possibility to

positively prove viral eradication in this patient.

In summary, our results demonstrate successful CD4+ T cell reconstitution at the systemic

level as well as in the largest immunologic organ following CCR5Δ32/Δ32 SCT, and

additionally provide evidence for the reduction in the size of the potential HIV reservoir over

time. Although the recovered CD4+ T cells are susceptible to infection with X4 HIV infection,

the patient remains without any evidence for HIV infection since more than 3.5 years after

discontinuation of ART. From these results, it is reasonable to conclude that cure of HIV

infection has been achieved in this patient.

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Acknowledgements

The authors are grateful to the patients for their participation in this project. We thank Diana

Bösel and Simone Spiekermann for excellent technical assistance, and Désirée Kunkel from

the Berlin-Brandenburg Center for Regenerative Therapies for technical support with cell

sorting. This work was supported by a research funding from the German Research

Foundation (DFG KFO104) to K.A. and T.S.. The HIV-1 molecular clone pNL4-3 from Dr

Malcolm Martin was provided by the EU Programme EVA Centre for AIDS Reagents,

NIBSC, UK (AVIP Cotract Number LSHP-CT-2004-503487). HIV-1 JR-CSF from Dr Isy

Chen was provided from the WHO-UNAIDS Virus Network through the Centre for AIDS

Reagents.

Authorship

Contribution: K.A. designed experiments; K.A., J.H., C.L. performed experiments and

analyzed data; K.A., C.L. made the figures; K.A., G.H., J.H., T.S. interpreted and discussed

the data; G.H., K.R., E.T. collected data; E.T. critically revised the manuscript for important

intellectual content; T.S. supervised the research; and K.A. wrote the manuscript. All authors

read and approved the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Kristina Allers, Department of Gastroenterology, Infectious Diseases, and

Rheumatology, Medical Clinic I, Campus Benjamin Franklin, Charité - University Medicine,

Hindenburgdamm 30, 12203 Berlin, Germany; e-mail: kristina.allers@charite.de.

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21

Table 1: Detection time points of HIV RNA and HIV DNA following CCR5Δ32/Δ32

SCT

HIV RNA

LTR and gag

(in months post-transplant)

HIV DNA

LTR and env

(in months post-transplant)

Plasma 0 – 45 (each month)

PBMC 0 – 45 (each month)

BMMCa 3, 12, 16.5, 40

CSF 14, 14.5, 15.5, 17

Brain 17

Colon

Mucosal CD4+ T cells

Mucosal macrophages

5.5, 24, 29

24, 29

24, 29

a

BMMC, bone marrow mononuclear cells

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22

Figure legends

Figure 1: Peripheral CD4+ T cells have been efficiently restored and contain an

increased proportion of activated/effector memory CD4+ T cells when

compared to healthy controls. CD4+ T cell numbers and frequencies of effector

memory cells (EM), central memory cells (CM), recent thymic emigrants (RTE), and

central naïve cells (CN) within CD4+ T cells (A) during the course of immune

reconstitution following CCR5Δ32/Δ32 SCT and (B) in SCT controls (27.5 ± 7 months

post-transplant) compared to healthy individuals were determined in fresh whole

blood. Median CD4+ T cell number of healthy individuals is indicated by the thick

horizontal line and the dashed horizontal lines denote the normal 25th and 75th

percentiles in (A). The horizontal lines in (B) denote the median values of each

group. Statistical significances are given for comparisons between healthy control

values and SCT control values (*P <>P <>P <>

expression of the activation markers CD38, HLA-DR and CD49d and the proliferation

marker Ki67 at 9.5 and 24 months after CCR5Δ32/Δ32 SCT in comparison to SCT

controls and healthy controls. Data are representative for five SCT controls and four

healthy controls.

Figure 2: The mucosal immune system has been efficiently repopulated with

donor-derived CD4+ T cells. (A) Immunohistochemical quantification of CD4+ T cells

in colon tissue of the CCR5Δ32/Δ32 SCT patient, SCT control patients (27 ± 9

months post-transplant) and healthy controls. The horizontal lines denote the median

values of each group. (B) Genomic DNA was extracted from mucosal CD4+ T cells

and subjected to CCR5-specific PCR spanning the Δ32 region.

Figure 3: CXCR4 surface expression on peripheral and mucosal CD4+ T cells is

not impaired in the CCR5Δ32/Δ32 SCT patient. CD4+ T cells in (A) fresh whole

blood, MMC (5.5 months post-transplant), or (B) ex vivo PHA/IL-2 activated PBMC

were analyzed for the frequency of CXCR4 surface expressing cells and the CXCR4

expression density. CXCR4 expression density on CD4+ T cells was evaluated as the

MFI of CXCR4 expression divided by the MFI value obtained with the corresponding

isotype control and is expressed as the MFI ratio.

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23

Figure 4: Recovered peripheral and mucosal CD4+ T cells are susceptible to

productive X4 HIV infection. PBMC (black symbols) and MMC (white symbols)

obtained 24 months after CCR5Δ32/Δ32 SCT were activated with PHA and IL-2 and

then incubated with the CCR5-tropic HIV-1 strain JR-CSF (triangles) or the CXCR4-

tropic HIV-1 strain NL4-3 (circles) at a MOI of 0.001. Viral replication was quantified

by measuring the amount of HIV core protein p24 in the cell-free supernatants of

cultures. No virus production was observed in the mock controls. Similar results were

obtained with peripheral lymphocytes purified at 9.5 and 34.5 and months after

CCR5Δ32/Δ32 SCT.

Figure 5: No evidence for residual HIV target cells of host origin in the liver and

the brain. CCR5-expressing CD4+ T cells or macrophages were detected (A) in liver

and (B) in brain tissue sections obtained 12 and 17 months after CCR5Δ32/Δ32 SCT,

respectively, by in situ immunofluorescence double staining for CD4 (green) or CD68

(green) and CCR5 (red). Original magnification x400. Images were acquired using

the AxioImager Z1 fluorescence microscope (Carl Zeiss MicroImaging, Jena,

Germany) coupled to the AxioCam MRm digital camera (Carl Zeiss). Acquisition

software: Axiovision (Carl Zeiss). Software used for image processing: Adobe

Photoshop CS (Adobe Systems, San Jose, CA).

Figure 6: Host macrophages were replaced with donor-derived cells during the

course of immune reconstitution. (A) CCR5-expressing macrophages were

detected by in situ immunofluorescence double staining for CD68 (green) and CCR5

(red) in colon tissue sections obtained 5.5 or 24 months after CCR5Δ32/Δ32 SCT.

CCR5-expressing macrophages are indicated by yellow arrows. (B) 24 and 29

months after CCR5Δ32/Δ32 SCT, macrophages were sorted from mucosal cells and

genotyped by CCR5 variant-specific PCR.

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Figures 1A and B

A

B

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Figure 1C

C

CCR5Δ32/Δ32 SCT SCT control healthy

9.5 months 24 months

For personal use only. by guest on December 11, 2010. www.bloodjournal.orgFrom

Figure 2

B

A

CCR5wt

CCR5Δ32300 bp

200 bp

Mucosal CD4+T cells

For personal use only. by guest on December 11, 2010. www.bloodjournal.orgFrom

Figure 3

CCR5Δ32/Δ32 SCT A CCR5 wild-type

Peripheral memory CD4+T

cells

Mucosal memory CD4+T cells

B

CCR5Δ32/Δ32 SCT CCR5 wild-type

PHA/IL-2 activated CD4+T cells

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Figure 4 For personal use only. by guest on December 11, 2010. www.bloodjournal.orgFrom

Figure 5

A

Liver

12 months

CD4 CD68

CCR5

DAPI

CCR5

DAPI

DAPICCR5

CD4

DAPICCR5

CD68

Brain

17 months

B

CD68

CCR5

DAPI

DAPICCR5

CD68

For personal use only. by guest on December 11, 2010. www.bloodjournal.orgFrom

Figure 6

CD68

CCR5

DAPI

DAPICCR5

CD68

CD68

CCR5

DAPI

DAPICCR5

CD68

A

Colon

5.5 months Colon24 months

B

Mucosal macrophages

CCR5wt

CCR5Δ32300 bp

200 bp

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Download links:

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Tip of the hat to the indefatigable Mr. Corporanon. Yet again.

Updates with links as I prepare them.

Please remember to respect the publisher's wishes : personal use only. Not for profit but for knowledge to we publish: long live the internet.

UPDATE:

Here you go, some links.

• That Doctor who said he could engineer a virus to use the human immune system against itself? His name was MacArthur: here's one available source for the info; links following.

Dr. Donald MacArthur, a high-level defense department biological research administrator, showed up at a June 9, 1969, meeting of a House subcommittee on military appropriations begging for cash to carry out an unsavory endeavor.

"Within five to ten years," he prognosticated, "it would provably be possible to make a new infective microorganism which would differ in certain important aspects from any known disease-causing organisms. Most important of these," he continued - and this is the ominous part, "is that it might be refractory to the immunological and therapeutic processes upon which we depend to maintain our relative freedom from infectious disease."

This new germ, the one Dr. MacArthur desired so sincerely to whip up in his lab, would destroy the immune system. The good doc proffered the most hackneyed of Cold War rationales for this odious ambition.

"Should an enemy develop it there is little doubt that this is an important area of potential military technological inferiority in which there is no adequate research program."

He got his coveted taxpayer funding. In 1977 and 1978, at the tail end of Dr. MacArthur's time frame, the first cases of Acquired Immune Deficiency Syndrome (AIDS) emerged in Africa.

Read More

The links for the above page.

• That legend about the clinics? It was Hep B, not C, otherwise its real all right. Here's a search on the topic; links are below it. That little unassuming graphic gets big when you click it. Links are here.

well besides that? More as I find it.

Be seeing you.

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