Gene Therapy (2006), 1–13
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ORIGINAL ARTICLE
T-cell protection and enrichment through lentiviral
CCR5 intrabody gene delivery
CH Swan1,3,4, B Bühler1,4, MP Tschan1, CF Barbas III2 and BE Torbett1,3
1
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; 2Department of Molecular
Biology, La Jolla, CA, USA and 3Department of Molecular Pathology, University of California, San Diego, La Jolla, CA, USA
CCR5 is the chemokine co-receptor for R5-tropic human
immunodeficiency virus type 1 (HIV-1) isolates most often
associated with primary infection. We have developed an
HIV-1 self-inactivating vector, CAD-R5, containing a CCR5
single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4+ T cells disrupts
CCR5 cell surface expression and provides protection from
R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody
expression in primary CD4+ T cells supports significant
growth and enrichment over time during HIV-1-pulsed
dendritic cell–T-cell interactions. These results indicate that
CCR5 intrabody-expressing CD4+ T cells are refractory
against this highly efficient primary route of infection.
CD34+ cells transduced with the CAD-R5 vector gave rise
to CD4+ and CD8+ thymocytes in non-obese diabetic (NOD)/
severely combined-immunodeficient (SCID)-human thymus/
liver (hu thy/liv) mice, suggesting that CCR5 intrabody
expression can be maintained throughout differentiation
without obvious cellular effects. CD4+ T cells isolated from
NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1
challenge demonstrating the maintenance of protection. Our
findings demonstrate delivery of anti-HIV-1 activity through
CCR5 intrabodies in primary CD4+ T cells and CD34+
cell-derived T-cell progeny. Thus, gene delivery strategies
that provide a selective survival and growth advantage
for T effector cells may provide a therapeutic benefit for
HIV-1-infected individuals who have failed conventional
therapies.
Gene Therapy advance online publication, 1 June 2006;
doi:10.1038/sj.gt.3302801
Keywords: HIV-1; HIV-1 vector; CCR5 intrabody; protection; enrichment; gene delivery
Introduction
A multiplicity of chemokine co-receptors have been
shown to facilitate human immunodeficiency virus type
1 (HIV-1) entry in tissue culture; however, only CCR5
and CXCR4 (the X4-tropic HIV-1 co-receptor) have been
convincingly demonstrated to be the relevant chemokine
receptors in man.1–4 Although viral isolates utilizing
CCR5 and CXCR4 for entry potentially could be
transmitted from individual to individual, it appears
that CCR5 using HIV-1 species are the predominant
viruses transmitted.5,6 Moreover, susceptibility to viral
infection and disease progression are correlated to cell
surface CCR5 levels. For example, individuals with a 32
base pair (bp) homozygous deletion in their CCR5 gene
(D32) lack functional CCR5 expression and are protected
against infection.7–10 Individuals heterozygous for the
D32 mutation have reduced levels of CCR5 and are
delayed in their progression to AIDS by 1–2 years.8
Additionally, mutations in the CCR5 promoter, such as
the 59029 G/A polymorphism, reduce activity by 45%
and also delay progression to AIDS by about 4 years.11,12
Correspondence: Professor BE Torbett, Department of Molecular
and Experimental Medicine, The Scripps Research Institute, University of California, San Diego MEM L55, 10550 North Torrey Pines
Road, La Jolla, CA 92037, USA.
E-mail: betorbet@scripps.edu
4
These authors contributed equally to this work.
Received 20 December 2005; revised 19 April 2006; accepted 20
April 2006
Finally, disruption of CCR5 expression does not appear
to be critical for normal cell function, as individuals
with these naturally occurring polymorphisms do not
seem to be associated with any detrimental phenotype
(see O’Brien and Moore4 for a review). Therefore,
intervention strategies aimed at altering CCR5 expression may be beneficial for cellular protection against
HIV-1 infection and provide a clinical benefit.
Specific targeting of chemokine receptors utilizing
intrakines, RNA interference (RNAi), intrabodies, or
ribozymes by vector delivery have proved effective in
tissue culture.13–16 However, these viral entry disruption
strategies have shown varying levels of protection when
the viral challenge dose is increased, thus providing the
caution that therapeutic efficacy may be breached
by high viral loads.17–19 Recent studies have indicated
that HIV-1 may be more effectively transmitted during
antigen presentation between CD4+ T cells and HIV-1
exposed or infected dendritic cells (DCs), termed an
infectious immunological synapse, thus providing a
means for increased local viral levels.20–24 As it may be
impossible to protect all susceptible cells in an individual
through gene delivery, the delivered protection must
function to allow a limited number of primary T cells
to survive, enrich, and function in the face of a robust,
widespread HIV-1 infection mediated via free virus and
cell-to-cell interaction.
Herein, we examined whether the HIV-1 derived selfinactivating (SIN) vector, CAD-R5, can deliver CCR5
intrabody genes to primary T and CD34+ hematopoietic
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
2
cells and whether the resulting intrabody expression in
CD4+ T cells promoted a survival and growth advantage
during HIV-1 challenge. The CCR5 intrabody is specific
for the N-terminal extracellular domain of CCR5 and
engineered with the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal.16,25 We determined that
expression of the CCR5 intrabody from the CAD-R5
vector resulted in almost complete disruption of CCR5
cell surface expression and protected both T-cell lines
and primary CD4+ T cells from robust infection with free
R5-tropic viruses. Furthermore, intrabody-expressing
CD4+ T cells, obtained from non-obese diabetic/severely
combined immunodeficient (NOD/SCID)-human thymus/liver (hu thy/liv) mice transplanted with CAD-R5transduced CD34+ cells, were protected from high-titer
R5-tropic HIV-1 challenge in tissue culture. Most
importantly, intrabody-expressing primary CD4+ T cells
have a selective survival and growth advantage during
DC-mediated viral challenge.
Results
CCR5 intrabody-mediated disruption of CCR5 cell
surface expression in THP-1 and primary CD4+ T cells
In our previous report, we demonstrated that a PM1
T-cell line expressing moderate levels of CCR5 intrabody
from cells with multiple transgenes was protected from
R5-tropic HIV-1 challenge generated by repeated transductions and drug selection.16 If cellular modifications of
chemokine receptors by gene delivery are to be effective
for abrogating HIV-1 entry in primary CD4+ T cells,
CCR5 intrabodies must function effectively when expressed from the gene delivery vehicle of choice at low
vector copy number. To determine whether CCR5
intrabodies can provide protection to leukocytes, lentiviral vectors were developed that expressed the CCR5
intrabody gene, CAD-R5, and a control vector, CAD,
which did not include the CCR5 intrabody gene (Figure
1a). To determine the efficacy of the CAD-R5 vector to
alter CCR5 expression, we first evaluated CAD-R5 vector
function in the THP-1 monocytic cell line, which is
known to express CCR5 at moderate to high levels on the
cell surface (Figure 1b).26 THP-1 cells were transduced at
a low multiplicity of infection (MOI ¼ 1) to obtain a low
vector copy number per cell. Two weeks after transduction, the mean fluorescence intensity (MFI) for CCR5
expression in the THP-1 CAD-R5 cell was decreased
from an MFI of 27–6, an expression level similar
to background levels obtained from the irrelevant (nonCCR5) isotype control antibody. These cells have been
maintained in tissue culture for 12 weeks with little
change in CCR5 or truncated nerve growth factor
receptor (tNGFR) expression (data not shown). These
findings show that CCR5 intrabody gene expression
from a lentiviral vector decreases both the percentage
of CCR5-positive cells and the overall amount of CCR5
available on the surface of each cell.
Next, we evaluated whether CCR5 intrabody gene
expression from the CAD-R5 vector would alter CCR5
expression on the cell surface of primary CD4+ T cells.
CD4+ T cells were transduced with CAD or CAD-R5
vectors, rested and reactivated to obtain maximum CCR5
expression levels.27,28 Consistent with our findings in
THP-1 cells, CCR5 expression levels in CAD-R5 vectortransduced primary CD4+ T cells shifted from an MFI
of 34 in the parental or control CAD vector-transduced
cells to background MFI levels of 8, an expression level
Figure 1 Evaluation of CCR5 intrabody function in the monocyte cell line THP-1 and in primary CD4+ T cells. (a) Vector design. The CADR5 HIV-1 SIN vector was used for CCR5 intrabody gene delivery. Vector elements are as follows: MND: myeloproliferative sarcoma virus
LTR-negative control region deleted; cPPT-CTS polypurine tract-central terminating sequence; cIRES: cellular internal ribosomal entry site;
tNGFR: truncated human nerve growth factor receptor; SAR: IFN-b-scaffold attachment region; and WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. The CAD vector does not contain the CCR5 intrabody gene, but all other elements, and as serves as a
control for intrabody function. (b) CAD-R5 vector expression and function in THP-1 cells. THP-1 cells transduced with CAD-R5 (thick line) or
CAD (thin line) vectors were evaluated by flow cytometry for relative CCR5 expression (see Materials and methods). Isotype antibody control
and flow cytometry analysis is shown as a dashed line. (CAD – 95% CCR5+, mean fluorescence intensity (MFI) ¼ 27; CAD-R5 – 9.7% CCR5+,
MFI ¼ 6). (c) CAD-R5 vector expression and function in primary CD4+ T cells. T cells transduced with the CAD-R5 vector (thick line)
demonstrated a fourfold reduction in CCR5 MFI (MFI ¼ 8) as compared to the CAD vector (thin line, MFI ¼ 34), as shown by flow cytometry
analysis for relative CCR5 levels. Isotype antibody control is shown as a dashed line. (d) CCR5 intrabody specificity in primary CD4+ T cells.
T cells transduced with the CAD-R5 vector (thick line) or the CAD vector (thin line) demonstrate comparable levels of CXCR4 by flow
cytometry. Isotype antibody control is shown as a dashed line.
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CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
similar to background levels obtained from the irrelevant
(non-CCR5) isotype control antibody (Figure 1c). Lastly,
this was a specific effect as CXCR4 expression levels
were not altered in CAD-R5-positive CD4+ T cells (Figure
1d). When taken together, these results are consistent
with the interpretation that the CAD-R5 vector significantly reduces CCR5 cell surface levels in primary CD4+
T cells.
CCR5 intrabody-expressing T-cell lines and primary
CD4+ T cells are resistant to R5-tropic HIV-1 challenge
To determine if the CAD-R5 vector provided protection
from R5-tropic HIV-1 challenge, PM1 T-cell lines were
established by selection of tNGFR reporter-expressing
CAD and CAD-R5 vector-transduced cells. The PM1
CAD and PM1 CAD-R5 lines, more than 99% positive
for the tNGFR, were challenged with 1% irradiated
R5-tropic NFN-SX-r-HSAS reporter-infected PM1 cells.
The NFN-SX-r-HSAS reporter virus expresses mouse
heat-stable antigen (mHSA) on the cell surface upon
productive infection allowing real-time flow cytometry
analysis of infected cells.16,29 Figure 2a presents the flow
cytometry analysis 6 days after the mixture of the
productively infected NFN-SX-r-HSAS cells with various
PM1 T-cell populations. The productively infected
parental PM1 line and CAD lines were readily identifiable as demonstrated by the right-shift of mHSA-positive
cells. In contrast, the PM1 CAD-R5 line remained mHSA
negative during challenge with R5-tropic chronically
infected PM1 cells. To evaluate nonspecific protective
effects of vector transduction and intrabody expression,
the PM1 CAD and CAD-R5 lines were infected with the
X4-tropic NL-r-HSAS mHSA-expressing reporter virus at
an MOI of 0.1. The PM1 parental, CAD and CAD-R5 cell
lines were mHSA positive following X4-tropic NL-rHSAS infection. These findings demonstrate the specificity of the CCR5 intrabody for altering CCR5 expression
and modifying R5-tropic infection and the absence of
nonspecific HIV-1-blocking effects from vector transduction and/or CCR5 intrabody expression.
Given the higher amounts of CCR5 present on primary
CD4+ T cells than PM1 T cells, we next evaluated whether
CAD-R5 vector transduction and CCR5 intrabody
expression provided protection from R5-tropic SF-162
viral challenge. Primary CD4+ T cells were transduced
with the CAD or the CAD-R5 vector, enriched for tNGFR
expression, cultured, reactivated after resting and then
challenged with SF-162 at an MOI of 1. Culture supernatants were collected for p24 analysis on various days
post-infection to determine HIV-1 production. The p24
results from two out of three representative experiments
demonstrate that the CAD-R5-transduced T cells are
resistant to HIV-1 infection and displayed a 50- to 60-fold
reduction (1.5 log) of p24 as compared to the CAD vector
and untransduced infected controls (Figure 2b). The
second experiment shown included untransduced control cells to confirm that cells transduced with the
CAD vector were able to obtain similar levels of HIV-1
infection as non-transduced cells. To determine the
specificity of CCR5 intrabody control and assess possible
global changes in viral infection brought about by
intrabody expression in primary CD4+ T cells, parental,
CAD and CAD-R5 stably transduced cells were challenged with the X4-tropic viral isolate laboratory adapted
isolates (LAI)/IIIB. As shown in Figure 2c, LAI enters
and replicates equally well in parental, CAD and CADR5 stably transduced cells. Together, the findings from
the PM1 and primary CD4+ T-cell HIV-1 challenge
studies demonstrate the therapeutic efficacy of CAD-R5
delivery and specificity of CCR5 intrabody-mediated
protection.
3
NOD/SCID-hu thy/liv reconstitution with CAD-R5transduced CD34+ cells
Our findings demonstrated consistent CCR5 intrabody
expression in cell lines and primary CD4+ T cells. To
evaluate whether CAD-R5 vector-transduced CD34+ cells
could give rise to thymocytes expressing functional
CCR5 intrabody, we utilized the NOD/SCID-hu thy/liv
mouse model.30–32 SCID-hu mice have been used
successfully to evaluate HIV-1 pathogenesis as well as
gene delivery to CD34+ cells and the subsequent
development to thymocytes.15,30,32–34 NOD/SCID mice
were implanted with human fetal liver–thymus–liver
sections under the kidney capsule to establish a co-joined
human fetal liver and thymus. CD34+ progenitor cells
were isolated from human leukocyte antigen (HLA)mismatched fetal liver cells and transduced with CAD or
CAD-R5 vector (two consecutive transductions at an
MOI of 25). Two months after the establishment of the
NOD/SCID-hu thy/liv mice, all mice were sub-lethally
irradiated to reduce endogenous human cells in the thy/
liv graft, and the next day 2.5 105 transduced HLAmismatched CD34+ cells (80% tNGFR positive) were
directly injected into the graft. Six weeks after reconstitution, the thy/liv grafts were harvested and analyzed
for human thymocyte development.
Table 1 presents the human CD4+ and CD8+ T-cell flow
cytometry profile for the HLA-mismatched donor population for all three experimental groups, mock transduced, CAD and CAD-R5 as a percentage of total donor
and NGFR-positive cells. Thymocytes from the CAD
and CAD-R5 NOD/SCID-hu thy/liv mice (n ¼ 2) were
positive for tNGFR reporter gene expression and both
displayed significant levels of tNGFR expression (15–
32%). All three groups displayed similar patterns of
thymocyte sub-populations with a majority of the cells
being CD4+/CD8+ and a small percentage of CD4+ and
CD8+ single positive cells. The CAD tNGFR-positive
population displayed a slightly higher percentage of
single positive CD4 and CD8 T cells as compared to the
CAD-R5 tNGFR population. However, this trend is seen
upon analysis of the whole HLA-mismatched population
for the CAD and CAD-R5 groups. These finding suggest
transduction, vector integration, CCR5 intrabody expression and/or CCR5 cell surface expression disruption did
not have an overt affect on thymocyte development.
CCR5 intrabody-mediated protection from R5-tropic
HIV-1 in primary thymocytes
As R5-tropic viral isolates are known to not replicate to
high levels and cause pathology in SCID-hu thy/liv
mice,33 we utilized a tissue culture assay for evaluating
CCR5 intrabody-mediated protection.34,35 To mimic
functionally an in vivo setting where not all CD4+ T cells
are protected during a viral infection, a mixture of
unprotected (non-transduced) and CCR5 intrabody gene-positive thymic-derived cells were used. We reasoned
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CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
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Figure 2 CCR5 intrabody-expressing cells are protected against R5-tropic, but not X4-tropic HIV-1 challenge. (a) Evaluation of CAD-R5
vector function in PM1 T cells challenged with R5-tropic or X4-tropic HIV-1. PM1 T cells were not transduced (Parental, left panels) or
transduced with CAD (CAD, middle panels) or CAD-R5 (CAD-R5, right panels) vectors. Cultures containing the various T-cell populations
were left uninfected (Uninfected, top panels), challenged with irradiated PM1 T cells (1% of total cells in culture) expressing the R5-tropic
reporter virus, NFN-SX-r-HSAS (+NFN-SX-r-HSAS, middle panels), or directly infected with the X4-tropic reporter virus (0.1 MOI) NL-rHSAS (+NL-r-HSAS, bottom panels). Both viruses express the mHSA on the cell surface upon productive HIV-1 infection. Parental, nontransduced PM1 T cells served as a positive control for susceptibility to infection. Six days after exposure to the reporter viruses, all PM1 cells
were assessed by flow cytometry for tNGFR and mHSA expression. (b) Evaluation of CAD-R5 vector function in primary CD4+ T cells when
challenged with R5-tropic HIV-1. T cells were not transduced (Control) or transduced with CAD (CAD) or CAD-R5 (CAD-R5) vectors. Some
CD4+ T-cell cultures were not infected (HIV) or infected with the R5-tropic SF-162 HIV at an MOI of 1 (+HIV) and viral replication was
determined at the times indicated by p24 evaluation. CCR5 intrabody-expressing CD4+ T cells showed 50- to 60-fold less virus as compared to
the CAD vector-expressing CD4+ T cells. Two results are shown from three representative experiments. (c) Evaluation of CAD-R5 vector
function in primary CD4+ T cells when challenged with X4-tropic HIV-1. Primary CD4+ T cells were not transduced (Control) or transduced
with CAD (CAD) or CAD-R5 (CAD-R5) vectors. Some CD4+ T-cell cultures were not infected (HIV) or infected with LAI at an MOI of 0.1
(+HIV) and viral replication was determined at the times indicated by p24 evaluation. All CAD or CAD-R5 CD4+ T-cells cultures were
composed of 498% tNGFR-expressing cells, see Materials and methods for additional information.
if CCR5 intrabody protection was evident, then viral
replication and spread might be predicted to be curtailed
in cultures containing CCR5 intrabody-expressing thymocytes given the reduced number of susceptible targets as
compared to cultures containing non-transduced or CAD,
vector control, transduced cells. To evaluate viral resistance, cultures were established with cells obtained from
thymi of control (mock transduced), CAD and CAD-R5
NOD/SCID-hu thy/liv mice. The CAD and CAD-R5
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thymocyte cultures were a mixture of 80% tNGFRpositive cells and 20% non-transduced cells obtained
from the magnetic bead separation column flow through.
Thymic cells in all cultures were activated for 3 days
with phytohemagglutinin (PHA), exposed to the R5tropic SF-162 HIV-1 (MOI 1) for 6 h on day 3, washed
and re-cultured. Viral replication over time was evaluated
by collecting tissue culture supernatants on various days
for p24 level determination.
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
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Table 1 Human thymocyte population analysis from NOD/SCID-hu thy/liv mice
HLA donor-specific total thymic population
Control
CAD (n ¼ 2)
CAD-R5 (n ¼ 2)
tNGFR-positive thymic population
CD4+/CD8+
CD4+/CD8
CD4/CD8+
tNGFR+
CD4+/CD8+
CD4+/CD8
CD4/CD8+
91
89
63
93
62
3
5
4
4
2
5
6
12
2
4
0
32
15
29
20
0
84
82
96
92
0
6
12
2
3
0
8
5
1
4
Abbreviations: HLA ¼ human leukocyte antigen; NOD/SCID-hu thy/liv mice ¼ non-obese diabetic/severely combined-immunodeficienthuman thymus/liver mice; tNGFR ¼ truncated nerve growth factor receptor. Values represent percentage of HLA donor-specific human cells
in the NOD/SCID-hu thy/liv implant.
In our viral challenge assays, differences were evident
in the amount and duration of viral replication in
cultures containing CAD-R5 intrabody-positive cells,
as compared to cultures containing CAD and non-transduced control CD4+ T cells. As can be seen in Figure 3,
p24 levels in the CAD-R5 cultures increased above the
uninfected control cultures until day 5 after which time
p24 levels remained constant throughout the 17-day
observation period, thus indicating that maximal viral
infection had been reached by day 5. In contrast, the
CAD and untransduced control cultures had increased
and continued viral replication, and presumably viral
spread, over the culture period as indicated by the
increasing p24 levels. Moreover, by day 17, the increased
viral replication in CAD and untransduced control
cultures resulted in approximately threefold higher p24
levels as compared to the CAD-R5 cultures. After day 11
in untransduced control cultures, the p24 levels fell,
presumably owing to loss of susceptible targets in
culture as the result of cellular death.
Enrichment of the PM1 CD4+ T-cell line expressing the
CCR5 intrabody upon R5-tropic HIV-1 infection
An underlying assumption for HIV-1 protective gene
therapy of CD4+ T cells is that the cellular protection
afforded by the delivered product will promote survival
and enrichment over time in the face of an ongoing
infection.36,37 Our previous findings using a neomycin
PM1 cell line selected for high levels of CCR5 intrabody
transgene integration demonstrated a 7-day survival
advantage after R5-tropic viral exposure.16,25 To further
investigate if CCR5 intrabody expression provides both
survival and cell expansion, we established assays to
determine if small numbers of PM1 CAD-R5-transduced
cells have a selective survival and growth advantage
during an ongoing HIV-1 infection.
PM1 T cells were transduced at a low MOI to achieve a
transduction frequency of less than 20% tNGFR-expressing cells. A low MOI was used to reduce the probability
of increased vector copy number per cell,38 thereby
allowing a determination of therapeutic efficacy at low
vector copy number. PM1 T-cell cultures were established with 15% CAD-transduced and 5% CAD-R5transduced PM1 cells, with the remaining cells in culture
being susceptible, parental PM1 T cells. To some cultures,
approximately 10% irradiated, NFN-SX-r-HSAS-infected
PM1 T cells were added. As all CAD- and CAD-R5-
Figure 3 CCR5 intrabody expression and function in thymocytes
derived from NOD/SCID-hu mice. Established human thymus/
liver implants in NOD/SCID-hu mice were injected with CD34+
HLA-mismatched mock-transduced cells or cells transduced with
the CAD or CAD-R5 vector. NOD/SCID-hu mice were allowed to
recover, and then thymi were harvested 6 weeks post-injection.
Thymocytes were collected and tNGFR-positive cells were isolated
by immunomagnetic bead selection from thymi reconstituted with
CAD or CAD-R5 vector-transduced CD34+ cells. Cultures were
established using mock- (Control), CAD vector- (CAD), or CAD-R5
vector- (CAD-R5) transduced thymic cells. The cellular composition
for the CAD-R5 vector cultures was 80% tNGFR-positive and 20%
non-transduced thymic cells to provide cells for viral replication.
Cultures were infected (+HIV) 3 days after activation with the R5tropic SF-162 virus at an MOI of 1. Viral replication was determined
at the time points indicated by p24 evaluation. The CAD-R5 vectortransduced T cells displayed a threefold p24 reduction as compared
to the CAD-transduced and -non-transduced T cells on day 17.
transduced cells express tNGFR, it is possible to follow
the survival and expansion of these cells by flow
cytometry and quantify changes over time. Moreover,
the number of virally infected PM1 T cells can be
determined by flow cytometry as well by using
antibodies to cell surface mHSA produced during a
productive NFN-SX-r-HSAS infection. Figure 4a displays
the outcome of these studies by showing the percentages
of tNGFR cells observed in cultures out to 15 days postinfection. The levels of tNGFR-positive uninfected PM1
CAD- and CAD-R5-transduced cells remained relatively
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CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
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Figure 4 CAD-R5 vector-transduced PM1 T cells increase over time in the presence of R5-tropic HIV-1 infection. (a) Flow cytometry analysis
of PM1 T cells stably expressing the CAD or CAD-R5 vector in the absence (HIV) or presence (+HIV) of HIV-1. Approximately 15% CAD
or 5% CAD-R5 vector-transduced PM1 T cells were co-cultured with 10% irradiated PM1 T cells not infected with NFN-SX-r-HSAS (HIV), or
infected with NFN-SX-r-HSAS (+HIV), with the remaining cells in the culture composed of parental PM1 T cells. Samples were removed at
5-day intervals from the cultures and evaluated by flow cytometry for the presence of tNGFR. Plotted are the percentages of cells expressing
tNGFR over time in each culture condition. By 15 days post-infection with NFN-SX-r-HSAS-infected cells, CAD-R5 vector-transduced PM1 T
cells enriched over 13-fold as compared to PM1 CAD-transduced T cells. (b) Assessment of HIV-1 replication in CAD-R5 vector-positive cells
grown in the presence of HIV-1-infected cells. To determine whether the tNGFR-positive PM1 CAD-R5 cells that grew out in the +HIV culture
displayed in (a) harbored virus, tNGFR-positive cells from the CAD-R5 +HIV group were isolated on day 21 of culture, thus isolating tNGFRpositive cells from the parental T cells, cultured for 6 additional days, and evaluated by flow cytometry for tNGFR- and mHSA-positive cells,
right panel, CAD-R5 +HIV. For comparison, flow cytometry analysis for the presence of tNGFR- and mHSA-positive cells from cultures of
CAD-R5 PM1 T cells grown in the absence of HIV, left panel, CAD-R5 –HIV, and parental PM1 T cells infected with and grown continuously
in the presence of HIV-1, middle panel, Parental +HIV, are shown.
constant throughout the experiment, thus showing no
competitive advantage for expansion over non-transduced PM1 T cells. In contrast, the percentage of tNGFRpositive PM1 CAD-R5 cells drastically increased in the
infected cultures to 78% of the total cell number by 15
days post-infection, Figure 4a, whereas the PM1 CAD
cells, in the presence of HIV-1-infected cells, failed to
increase over time, but were infected as determined by
p24 expression (data not shown). Thus, only the CAD-R5
intrabody containing PM1 T cells increased over time.
To determine the HIV infection status of CAD-R5transduced PM1 T cells that increased in HIV-positive
cultures, all cultures were maintained till day 21, and
then tNGFR-positive cells present in the CAD-R5 +HIV
culture group were isolated by two rounds of magnetic
bead separation (X99% tNGFR positive after separation,
data not shown), and then cultures were re-established.
The use of magnetic bead separation allowed isolation of
tNGFR-positive cells, regardless of infection status, from
the remaining non-tNGFR-positive infected parental
cells in the tissue culture mixture. As controls, the
cultures of chronically HIV-infected parental PM1 T cells
and PM1 T cells from the uninfected, CAD-R5 cultures
were maintained as well. After 6 days of additional
tissue culture, 27 days from the start of the original
cultures, cell samples from all three cultures were
evaluated for tNGFR and mHSA expression by flow
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cytometry. The findings are presented in Figure 4b. The
panel on the left demonstrates maintenance in tissue
culture of low numbers of tNGFR-positive CAD-R5 PM1
T cells in the absence of viral infection. The middle panel
shows the pattern of continued and chronic infection of
parental PM1 T cells, with some cells possibly becoming
latently infected as reflected by loss of mHSA expression.
The right-most panel presents the findings of CAD-R5
tNGFR-positive cells, isolated from HIV-1-infected cultures, after 6 days in tissue culture. As can be seen, there
is little indication of mHSA-positive cells (p1%) by flow
cytometry. Thus, it appears that tNGFR-positive cells
isolated from HIV-1-infected cultures after 21 days and
cultured for 6 additional days contained few infected
cells. It should be noted that the p1% mHSA-positive
CAD-R5 PM1 T cells obtained from the day 6 culture
might indicate that a small number of tNGFR-positive
cells were productively infected or that tNGFR-negative
cells were carried along during magnetic bead separation. Alternatively, this observation could be owing to the
uptake of the GPI-linked mHSA by uninfected, tNGFRpositive cells in the original 21-day cultures. To distinguish between the possibilities of continued infection or
mHSA carryover, the CAD-R5 PM1 T-cell cultures after
27 days were maintained for an additional 5 weeks and
evaluated by flow cytometry. The long-term cultured
tNGFR-positive cells were found to be p1% mHSA
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
7
positive by flow cytometry (data not shown). These
results suggest that if infected cells were present in the
culture, they died off or were overgrown by non-infected
cells over time, or that the mHSA reactivity was a carry
over from the original isolation. Overall, our findings
suggest that the majority of PM1 T cells expressing CCR5
intrabody, which survive, grow and expand during
a 21-day culture in the presence of infected PM1 T cells,
are mHSA negative and remain so when isolated from
culture.
Primary T cells expressing CCR5 intrabody are
protected from DC-mediated HIV-1 infection and
enrich over time
Cell-to-cell contact, specifically between HIV-1-infected
or viral-positive professional antigen-presenting cells
and uninfected CD4+ T cells in lymph nodes and other
immune privilege sites, termed the infectious immunological synapse, has been proposed to be a highly
efficient and, most likely, a major physiological means
of CD4+ T-cell infection.20–24 As we have demonstrated,
CAD-R5-transduced primary CD4+ T cells were refractory to infection by free virus. However, given the major
role of DCs in HIV-1 T-cell infection, we considered it
necessary to evaluate whether CAD-R5-transduced
primary CD4+ T cells are protected and expand when a
stronger and more efficient HIV-1 challenge is mediated
through viral presentation by DCs. To accomplish this
goal, we used a modified version of the assays developed
by Gummuluru et al.22,39 This tissue culture method
relies on HIV-1-pulsed DCs to form an immune synapse
with CD4+ T cells, allowing for a productive cell-to-cellmediated infection.20,24 Activated, primary CD4+ T cells
were transduced with either the CAD or CAD-R5 vector
at low MOI to achieve 20–30% transduction efficacy,
cultured, rested, reactivated and equal numbers of CADor CAD-R5-transduced cells were then co-cultured with
autologous, DCs that have been pulsed with R5-tropic
SF-162. Figure 5 presents the results of the changes of
tNGFR-positive cells for two independent studies. Flow
cytometry analyses before DC introduction on day 0, and
on days 6 and 9 post-dendritic co-culture demonstrated
a significant amount of enrichment of the CAD-R5transduced CD4+ T cells. In two independent experiments, CD4+ T cells transduced with the CAD-R5 vector
enriched by 75 and 67%, respectively, by day 9. In
contrast, T-cell cultures containing the CAD-transduced
CD4+ T cells decreased 31 and 35% in tNGFR-positive
cells. These results suggest that CAD-R5-transduced
CD4+ T cells have a survival and/or growth advantage
in the presence of an ongoing DC-mediated HIV-1
infection.
Discussion
Small-molecule chemokine receptor antagonists have the
potential to disseminate throughout the patient to most
immune compartments and disrupt HIV entry.40,41 In
contrast, clinical gene delivery at the current time is
limited in its ability to target most HIV susceptible cells
in patients.36 An underlying rationale for clinical use of
anti-HIV gene delivery is that susceptible cells expressing the protective gene of interest will be refractory
to viral infection, survive and enrich during an immuno-
Figure 5 CAD-R5 vector-transduced primary CD4+ T cells increase
over time during DC-mediated R5-tropic HIV-1 challenge. CAD and
CAD-R5 vector-transduced CD4+ T cells were co-cultured with
R5-tropic SF-162 pulsed DCs (+HIV) or DCs not pulsed with virus
(HIV) at a ratio of 1:10 DCs:CD4+ T cells. CD4+ T cells were
composed of 20–30% CAD or CAD-R5 vector-positive, tNGFRexpressing cells, with the remaining cells not transduced with
vector. Cultures were evaluated by flow cytometry for changes in
the numbers of CD4+, tNGFR-positive T cells before DC introduction, day 0, and after the introduction of DCs on days 6 and 9.
Plotted are the changes in the percentages of tNGFR-positive cells
over time in each culture condition.
logical response to HIV infection. Surprisingly, only a
few anti-HIV gene delivery strategies have been evaluated for their ability to provide a survival and
enrichment function in the presence of an ongoing HIV
infection.19,42,43 Our findings demonstrate that HIV-1
vector delivery of CCR5 intrabody genes to T-cell lines
and primary CD4+ cells rendered them resistant to HIV-1
challenge, either from free or DC presented virus. Most
importantly, CCR5 intrabody-expressing T cells have
a competitive survival and growth advantage during
ongoing HIV-1 viral spread in tissue culture.
The introduction of highly active anti-retroviral
therapy has resulted in clinical management of HIV-1
infection and has changed the course of a fatal disease to
one that is now a chronic condition.44,45 However, antiretroviral drug toxicity and the lack of patient compliance have given rise to drug-resistant viral mutations,
viral progression and the urgent need for new anti-viral
treatments.46–49 In this regard, CCR5 is an attractive
target for therapeutic intervention to halt viral entry, as
CCR5-dependent virus is associated with primary and
early stages of infection,6 and individuals with a
homozygous mutation of CCR5 (D32) are resistant to
R5-tropic HIV-1 infection.7–10,50 Currently, there are
a number of chemokine receptor antagonists under
review.40,41,51–53 An alternative therapeutic method for
disrupting HIV entry is the use of gene delivery to
provide a genetic means to alter expression or block the
function of chemokine receptors, for example, intrabodies, ribozymes, intrakines, zinc-fingers or RNAi.13–16,54
Our approach to disrupting CCR5 expression on the cell
surface relies on cellular expression of a humanized
intrabody targeting CCR5.16 The CCR5 intrabody recogGene Therapy
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
8
nizes and binds the sequence YYTSEP in the first
extracellular domain of CCR5 at low nM affinity.55 The
intrabody binding motif partially overlaps with the
gp120 binding and fusion motif.56–60 To further improve
cellular efficacy of the intrabody, a KDEL endoplasmic
retention signal has been included, which provides
entrapment of newly synthesized and recycled CCR5 as
well and maintains cellular compartmentalization of the
CCR5 intrabody.16,25 As shown in Figure 1b and c, CCR5
levels are substantially reduced (X95%) in lymphocyte
and myeloid cell lines and, most importantly, primary
CD4+ T cells. Moreover, the CCR5 intrabody targeting
effect is restricted to CCR5 as seen in the lack of CXCR4
disruption, Figure 1d, and protection to X4-tropic
viruses, Figure 2c.
Direct HIV challenge experiments at relatively high
input (MOI 1) provide evidence that humanized CCR5
intrabody expression from stable integration of the CADR5 vector in primary CD4+ T cells was sufficient to
disrupt CCR5 and prevent infection (Figure 2b). Moreover, CCR5 intrabody expression in a small number of
PM1 T cells allowed survival and enrichment over time
in infected cultures, further confirming that potent cellto-cell viral spread to CCR5 intrabody-expressing cells
was curtailed (Figure 4a). Moreover, conformation that
CAD-R5-positive T cells remained for the most part
mHSA negative for 21 days while co-cultured with HIV1-infected T cells further supports the notion that CCR5
levels were insufficient for viral entry (Figure 4b). Recent
studies by Cordelier et al.18 using SV40 delivery of CCR5
intrabody genes to cell lines and primary monocytes
were found to be susceptible to HIV infection despite
showing substantial reduction in CCR5 expression.
However, therapeutic efficacy could be achieved if a
second SV40 vector containing a CCR5 ribozyme was cotransduced, thus suggesting that an additional genetic
disruption mechanism was required to lower levels of
cell surface CCR5 to some threshold, thereby abrogating
infection. The concern of partial protection from viral
infection has been noted for CCR5 disruption mechanisms when used individually to protect T cells, such as
short hairpin RNA, ribozymes or intrabodies, when
higher viral challenge amounts were used.17,18,61 A
possible explanation for the enhanced efficacy of the
CCR5 intrabody in our studies could lie in the coupling
of the KDEL sequence with a high-affinity intrabody.16,25
The use of the KDEL allows efficient intrabody trapping
of both recycled and newly expressed receptor in the
endoplasmic reticulum, thereby enhancing CCR5 loss at
the cell surface.
An advantage of HIV-1-derived vectors is their ability
to transduce both dividing and non-dividing cells such
as resting T cells, DCs, macrophages and non-cycling
hematopoietic stem cells (HSCs), thereby limiting problems associated with HSC culture, such as maturation
and loss of stem cell potential.62,63 Our findings from
CD34+ cell transplant studies in NOD/SCID-hu thy/liv
mice suggest that CAD-R5 vector and CCR5 intrabody
expression does not overtly alter T-cell development
(Table 1). Furthermore, intrabody expression is stable
and remains functional for at least 8 weeks. These results
are in accord with our observations that CAD- and CADR5-transduced primary and T-cell lines did not demonstrate discernable alterations in growth as compared to
untransduced cells (data not shown). Additionally, we
Gene Therapy
have observed long-term intrabody expression, without
intrabody silencing or cellular alterations, in human
B- and myeloid cell populations obtained from long-term
repopulating cells in NOD/SCID mice transplanted with
CAD and CAD-R5 vector-transduced CD34+ cells (KL
Fischer and BE Torbett, unpublished studies, 2005). The
use of intrabodies in other cell-based systems have not
been reported to alter growth and development.64–66
Although CCR5 intrabody expression is functional and
limits the amount and duration of viral spread at the
population level in CD4+ T cells isolated from the human
thymus (Figure 3), additional studies are required to
determine the variability of CAD-R5-mediated protection provided to individual CD4+ T cells derived from
transduced CD34+ progenitors.
DCs are the pivotal antigen-presenting cells for
induction of T-cell immunity to pathogens and recent
evidence suggests that HIV-1 preferentially targets CD4+
T cells specific for HIV-1 antigens in infected individuals.67 Additional evidence points to enhanced viral
replication in DC–CD4+ T-cell clusters and the highly
efficient ability of DCs to transfer virus to susceptible
cells in the immunological synapse.20–24 We found
that CCR5 intrabody-expressing primary CD4+ T cells
increase during co-culture with viral-pulsed DCs, as
compared to reporter vector control CD4+ cells, which
implies a competitive advantage for cells having reduced
CCR5 expression (Figure 5). The primary CD4+ T-cell
enrichment results, when taken with the findings of the
CCR5 intrabody protection in direct viral challenges,
indicates that delivery of CCR5 intrabody genes allows
survival and protection from robust viral challenges.
These results further imply that we have achieved
therapeutic efficacy in small numbers of CD4+ T cells in
a population setting by decreasing the threshold of CCR5
available for viral entry. We propose that there may be an
advantage to removing CCR5 from the cell surface via
gene delivery, in contrast to using CCR5 small molecule
antagonists or gene delivery methods that block CCR5
on the cell surface, to reduce the probability of selecting
viral mutants that would continue to use CCR5 for viral
entry in the presence of the antagonist.68,69
The consequence of reducing the level of CCR5, as to
whether the CCR1 and CCR3 receptors will compensate
for the loss of CCR5 in vivo, remain unknown. Furthermore, a report by Glass et al.70 have determined that the
CCR5 D32 deletion is significantly related to symptomatic and fatal consequence of West Nile virus infection.
It is therefore necessary to examine the full spectrum of
immunological defects that may arise by disrupting
CCR5 in HSCs and T cells in large animal models.
In summary, our studies provide evidence that CCR5mediated cell surface disruption by HIV vector expression of CCR5 intrabodies both protects and allows
expansion of primary CD4+ T cells. Coupled with HIV
vector delivery, intrabody targeting of chemokine receptors provides a powerful phenotypic disruption strategy
possibly suitable for HIV-1 salvage therapy.
Materials and methods
Primary T and hematopoietic cells
Whole-blood samples were obtained from healthy
donors under the auspices of the General Clinical
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
Research Center at The Scripps Green Hospital, La Jolla,
CA, USA. Human fetal liver and thymus tissue were
obtained from fetuses at 16–24 weeks gestation (Advanced Bioscience Resource Inc. Oakland, CA, USA).
Protocols and the use of all human samples were
approved by the International Review Board of The
Scripps Research Institute, La Jolla, CA, USA.
Peripheral blood lymphocytes (PBLs) were isolated
from blood via density-gradient centrifugation using
Ficoll–Hypaque Plus (Amersham Biosciences, Sweden).
CD8+ cells were removed by positive selection using
anti-CD8+ magnetic microbeads (Stem Cell Technologies,
Canada) and a Miltenyi magnetic column (Miltenyi
Biotech, Germany) as per the manufacturer’s protocol.
Purity was assessed by flow cytometry with the appropriate chromophore-labeled antibody reagents. CD34+
fetal liver cells were selected using anti-CD34+ magnetic
microbeads (Stem Cell, Canada) and isolated on a
Miltenyi magnetic column (Miltenyi Biotech, Germany)
as described.62
Cell culture
293 T cells (a kind gift of CellGenesys, San Francisco, CA,
USA) were cultured in Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum (FBS),
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
10 mM
acid (HEPES) and antibiotics (GIBCO/BRL, Invitrogen,
Carlsbad, CA, USA), and maintained in a humidified
371C incubator containing 10% CO2.
PM171 and THP-172 cells were cultured in RPMI 1640
containing 10% FBS, 10mM HEPES and antibiotics
(cRPMI), and passaged every 3 days. All cell lines were
maintained in a 371C humidified incubator containing
5% CO2.
For primary CD4+ T-cell studies, 1 106 CD8+depleted PBLs/ml were cultured in cRPMI and activated
with 5 mg/ml PHA and 20 U/ml interleukin (IL)-2. After
2 days of culture, cells were washed and cultured in
cRPMI with 20 U/ml IL-2. Cells were reactivated on day
9 of culture with 5 mg/ml PHA and 20 U/ml IL-2 for 2
days. After 2 days of reactivation, cells were cultured in
cRPMI with 20 U/ml IL-2. All cultures were maintained
in a 371C humidified incubator containing 5% CO2.
CD34+ cells isolated from the fetal liver were cultured
at 4.5 106 cells/ml in Iscove’s modified Dulbecco’s
medium, 10% bovine serum albumin, insulin and
transferrin (BIT) (Stem Cell, Canada), 1% FBS, 50 ng/ml
thrombopoietin, 50 ng/ml stem cell factor, 20 ng/ml
IL-6, 100 ng/ml FLT-3, antibiotics and 1 mM b-mercaptoethanol for vector transduction purposes. All cultures
were maintained in a 371C humidified incubator containing 5% CO2.
HIV-1 production
The R5-tropic NFN-SX-r-HSAS and X4-tropic NL-rHSAS mHSA reporter viruses were prepared by transfection of the molecular clone into 293 T cells to generate
infectious virus, which was then amplified by infecting
the PM1 T-cell line or primary cells.16,29 NHL-r-HSAS is
based on NL4-3 with the murine heat-stable antigen in
place of vpr.16,29 NFN-SX-r-HSAS use the env of JR-FL,
which replaces the endogenous env of NHL-r-HSAS.16,29
The R5-tropic SF-162 and the X4-tropic LAI viruses were
obtained from viral supernatant infection of PHA (5mg/
ml)-activated primary CD8+-depleted PBL cultures.
Tissue culture supernatant was collected 7–9 days postculture and tested for p24 concentration, by the
University of California, San Diego, CA, USA, CFAR,
using the antigen capture ELISA test (Beckman Coulter,
Fullerton, CA, USA) according to the manufacturer’s
instructions.
9
Generation of the CAD and CAD-R5 HIV-1 SIN vectors
To ensure long-term expression in hematopoietic cells,
we have generated an HIV-1 SIN vector, which incorporated a number of unique elements (Figure 1a, CAD
series vector). A myeloproliferative sarcoma virus longterminal repeat (LTR)-negative control region deleted
(MND, myeloproliferative sarcoma virus LTR-negative
control region deleted) was selected as the internal
promoter based on the demonstration of high expression
of selected genes (unpublished data and Halene et al.73
and Aviles Mendoza et al.74) and the presence of altered
LTR elements that preclude methylation, which supported long-term expression in mouse long-term hematopoietic progenitors upon repeated passage.62,75 Lastly,
we have also included the 800 bp 30 interferon (IFN)-bscaffold attachment region (SAR) element 30 of all genes.
A SAR element may be critical for keeping the vector
transcriptionally active during the resting state of a cell,
after cell expansion and for inhibiting methylation.76–78
When present in an MLV vector, a SAR element has been
shown to increase expression in both resting and
activated human T and myeloid cells and provides
protection from silencing.76–78
A complete description of the CAD vector cloning
strategy is available upon request. Briefly, the CAD
control vector was assembled by standard cloning
techniques from the following elements: vector backbone
was derived from CS-PRE, a derivative of HIV-CS (kind
gift of H Miyoshi,75 University of Tsukuba, Japan), which
has unique BamHI, SacII, EcoRI, XbaI, HpaI and XhoI sites
upstream of the post-translational regulatory element.79
The MND LTR was cloned from the MND-HSPSV-EGFP
plasmid (kind gift of Don Kohn, USC School of Medicine,
Children’s Hospital Los Angeles, Los Angeles, CA, USA)
and cloned into the BamHI site. The 178-bp polypurine
tract-central terminating sequence (cPPT-CTS) BamH1/
Sma1 fragment was amplified from HIV-1 molecular
clone R8.80 The cellular internal ribosomal entry site
(cIRES) and tNGFR (cIRES-tNGFR) BamHI/ClaI fragment were cloned from plasmid pSK+100eIF4 (kind gift
of Gabor Veres, SyStemix, Palo Alto, CA, USA). The
800 bp 30 SAR element was cloned from plasmid pCL,
introducing Xba1/Hpa1 sites.81 The humanized CCR5
intrabody, ST6/34, was generated and cloned as
described previously.55 The CAD-R5 CCR5 intrabody
vector was constructed from CAD by replacing the
BamHI MND promoter fragment with an MND promoter
and the intrabody fragment.
CAD and CAD-R5 virion particles were produced
by transient calcium phosphate co-transfection as
described.62,75 Briefly, the CAD and CAD-R5 transfer
vectors (10 mg/dish) were co-transfected with the packaging construct pMDLg/pRRE (6.5 mg), pRSV-rev (2.5 mg)
and a vesicular stomatitis virus-G expression construct
(3.5 mg) into 2 106 293 T cells.62 Tissue culture supernatant was harvested 24 and 48 h later and concentrated
1000-fold by ultracentrifugation. MOI was calculated
based on serial titers using 293 T cells in triplicate as an
Gene Therapy
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
10
indicator line, as we have previously described in
Miyoshi H et al.62,75
HIV-1 vector transduction
PM1 and THP-1 cells were transduced in the presence of
8 mg/ml polybrene or protamine sulfate, respectively,
in cRPMI with selected MOI of 1 CAD or CAD-R5 vector
overnight, washed and maintained as described above.
CD8+-depleted PBLs were transduced with the CAD or
CAD-R5 vector at an MOI of 50 on day 2 of culture for
8 h in the presence of 8 mg/ml polybrene. Cells were
washed and cultured in complete RPMI with 20 U/ml
IL-2. Seven days post-transduction, tNGFR-positive cells
were isolated via immunomagnetic bead isolation using
NGFR-phycoerythrin (PE)-conjugated antibody (BD
Biosciences: Pharmingen, San Diego, CA, USA) and
a-PE dextran/a-dextran magnetic beads (Stem Cell Technologies, Canada) using a Miltenyi magnetic column
(Miltenyi Biotec, Germany). Isolated cells were reactivated with 5 mg/ml PHA and 20 U/ml IL-2 for 2 days,
and then cultured in cRPMI with 20 U/ml IL-2.
CD34+ fetal liver stem cells were transduced with the
CAD or CAD-R5 vector (MOI 25) on days 2 and 3 of
culture overnight in the presence of 3 mg/ml polybrene.
Cells were washed after each transduction and maintained as described above.
HIV-1 infection
PM1 cells were infected with R5-tropic NFN-SX-r-HSAS
HIV-1 and lethally irradiated and used to challenge the
PM1 T-cell line. As a CCR5 intrabody specificity control,
PM1 cells were infected with X4-tropic NL-r-HSAS at an
MOI of 0.1. Primary T cells were infected with R5-tropic
SF-162 (MOI 1) or X4-tropic LAI (MOI 0.1) HIV-1 for 3–6
hours in the presence of 8 mg/ml polybrene on day 11
of culture, cells were washed and re-cultured for the
desired time. Tissue culture supernatant was collected
during various time points for HIV-1 p24 determination
and p24 was determined, using triple or quadruplicate
samples, by the University of California, San Diego, CA,
USA, CFAR, using the antigen capture ELISA test
(Beckman Coulter) according to the manufacturer’s
instructions. All p24 amounts are presented as
mean7s.d., where appropriate. In many cases, the
variation was less than 10% of the mean.
Flow cytometry analysis
CAD and CAD-R5 tNGFR reporter gene expression
was detected by flow cytometry analysis on cells stained
with the PE-labeled antibody C40-1457 (BD Biosciences:
Pharmingen, San Diego, CA, USA) to NGFR. The
fluorescein isothiocyanate (FITC)-labeled version was a
kind gift of Gabor Veres, SyStemix (Palo Alto, CA, USA).
NFN-SX-r-HSAS and NL-r-HSAS HIV reporter gene
expression on the cell surface was detected by the
FITC-labeled antibody H1/69 to mHSA (eBioscience, San
Diego, CA, USA). CCR5 expression on primary T cells
was detected by indirect staining with 2D7 antibody (BD
Biosciences: Pharmingen, San Diego, CA, USA) followed
by PE-labeled donkey anti-mouse serum (Jackson Immunoresearch, West Grove, PA, USA) or directly using
PE-labeled 2D7 antibody (BD Biosciences: Pharmingen,
San Diego, CA, USA). CCR5 expression on THP-1 cells
was detected by PE-labeled 2D7 antibody. CXCR4
Gene Therapy
expression on primary T cells was detected by PE- or
FITC-labeled 12G5 antibody (BD Biosciences: Pharmingen, San Diego, CA, USA). Cells were analyzed on
Becton Dickinson FACScan using CELLQUEST software.
NOD/SCID-hu thy/liv mouse model
NOD/SCID mice were bred and treated with antibiotics
in accordance with the animal care and use committee at
The Scripps Research Institute (ARC no. 06JAN02). Mice
(7–8 weeks old) were placed under anesthesia (methoxyflurane and pentobarbitol) during the surgical procedure. Human fetal liver–thymus–liver tissue sections
(B3 mm3) were placed under the left kidney capsule.
Two months post-thymus establishment, mice were
irradiated with 325 rads. One day post-irradiation, mice
were placed under anesthesia as described above and
2.5 105 CD34+ HLA-mismatched fetal liver cells (mock,
CAD- or CAD-R5-transduced cells) were injected into the
human thymus. Six weeks post-CD34+ injection, mice
were killed and thymi were harvested for analysis and
prepared into a single-cell suspension for flow cytometry
analysis and cell culture.
Two-color flow cytometry analysis was performed on
thymocytes stained with anti-human antibodies, CD4+FITC and CD8+-PE (Beckman Coulter, Fullerton, CA,
USA), HLA-A2 FITC, CD8+-FITC and NGFR-PE (BD
Biosciences, Pharmingen, San Diego, CA, USA). The
CAD and CAD-R5 thymocyte groups were enriched for
tNGFR-positive cells via immunomagnetic isolation as
described above. Cultures were established with 80%
tNGFR-positive thymocytes and 20% non-transduced
thymocytes and activated with 5 mg/ml PHA and
20 U/ml IL-2 for 3 days in cRPMI, washed and cultured
in cRPMI with 20 U/ml IL-2. On day 3 of culture, cells
were infected with R5-tropic SF-162 at an MOI of 1 in the
presence of 8 mg/ml polybrene, washed and re-cultured
for the desired time. Tissue culture supernatant was
collected during various time points for HIV-1 p24
concentration and prepared as described above.
Primary CD4+ T-cell enrichment assay
The primary T-cell enrichment assay is a modification of
the rapid-turnover assay developed and described by
Gummuluru et al.22,39 CD8+ T cells were isolated and
depleted as described previously. Immature DCs were
enriched from an autologous donor via immunomagnetic cell sorting using anti-CD14+ microbeads (Stem
Cell Technologies, Canada) and isolated on a Miltenyi
magnetic column (Miltenyi Biotech, Germany). CD4+ T
cells were activated, cultured and transduced with the
CAD and CAD-R5 vector as described previously. DCs
were cultured at 1 106 cells/ml in cRPMI plus 500 U/
ml IL-4 and 800 U/ml granulocyte–macrophage colonystimulating factor (GM-CSF) for 7 days. On day 7 of
culture, DCs were pulsed with 500 ng p24 SF-162 viral
supernatant for 2–6 h and washed thoroughly three
times with phosphate-buffered saline. The HIV-1exposed DCs were then co-cultured with the CAD- or
CAD-R5-transduced CD4+ T cells at a ratio of 1:10 with
tNGFR-positive transduced cells representing 20–30% of
the total cell population. CD4+ T cells were reactivated
for 2 days with 5 mg/ml PHA and 20 U/ml IL-2 before
co-culturing with DCs. CD4+ T cells and DCs were cocultured in cRPMI with 20 U/ml IL-2, 500 U/ml IL-4 and
800 U/ml GM-CSF, and media were changed every 3
CCR5 intrabody-mediated protection and enrichment of T cells during HIV-1 infection
CH Swan et al
days. Cells were stained with NGFR-PE before the
introduction of DCs on day 0, and on days 6 and 9 post
co-culture for flow cytometry analysis. Enrichment was
determined by the percentage of tNGFR-positive cells.
Acknowledgements
We thank Leslie Romero and Laura Crisa, PhD, MD,
TSRI, for assistance with the NOD/SCID-hu thy/liv
surgeries and mouse maintenance, Mike McCune, MD,
PhD, Cheryl Stoddart, PhD, and Jose Rivera, UCSF, for
SCID-hu training, and Jerry Zack, PhD, and Beth
Jamieson, PhD, UCLA, for mHSA viruses. We are
grateful for the support of the late Dr Nava Sarver. We
also thank the UCSD CFAR (5P30 AI36214) for p24
determination. BB was supported by a Fellowship from
UARP F00-SRI-036. This research was supported by
NIH/NAID AI49165, AI40882 and AI29329-16 (BET) and
GM065059 (CFB). This is publication 17294-MEM from
The Scripps Research Institute.
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