Article

Yukl, SA, Shergill, AK, McQuaid, K, Gianella, S, Lampiris, H, Hare, CB et al.. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS 24: 2451-2460

San Francisco VA Medical Center (SFVAMC) and University of California, San Francisco (UCSF), San Francisco, California, USA.
AIDS (London, England) (Impact Factor: 5.55). 10/2010; 24(16):2451-60. DOI: 10.1097/QAD.0b013e32833ef7bb
Source: PubMed

ABSTRACT

To determine whether raltegravir-containing antiretroviral therapy (ART) intensification reduces HIV levels in the gut.
Open-label study in HIV-positive adults on ART with plasma HIV RNA below 40 copies/ml.
Seven HIV-positive adults received 12 weeks of ART intensification with raltegravir alone or in combination with efavirenz or darunavir. Gut cells were obtained by upper and lower endoscopy with biopsies from duodenum, ileum, colon, and rectum at baseline and 12 weeks. Study outcomes included plasma HIV RNA, HIV DNA and RNA from peripheral blood mononuclear cells (PBMC) and four gut sites, T-cell subsets, and activation markers.
Intensification produced no consistent decrease in HIV RNA in the plasma, PBMC, duodenum, colon, or rectum. However, five of seven participants had a decrease in unspliced HIV RNA per 10 CD4(+) T cells in the ileum. There was a trend towards decreased T-cell activation in all sites, which was greatest for CD8(+) T cells in the ileum and PBMC, and a trend towards increased CD4(+) T cells in the ileum.
Most HIV RNA and DNA in the blood and gut is not the result of ongoing replication that can be impacted by short-term intensification with raltegravir. However, the ileum may support ongoing productive infection in some patients on ART, even if the contribution to plasma RNA is not discernible.

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Available from: Huldrych F Günthard
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Effect of raltegravir-containing intensification on HIV
burden and T-cell activation in multiple gut sites of
HIV-positive adults on suppressive
antiretroviral therapy
Steven A. Yukl
a
, Amandeep K. Shergill
a
, Kenneth McQuaid
a
,
Sara Gianella
b
, Harry Lampiris
a
, C. Bradley Hare
c
, Mark Pandori
d
,
Elizabeth Sinclair
c
, Huldrych F. Gu¨nthard
b
, Marek Fischer
b
,
Joseph K. Wong
a
and Diane V. Havlir
c
Objective: To determine whether raltegravir-containing antiretroviral therapy (ART)
intensification reduces HIV levels in the gut.
Design: Open-label study in HIV-positive adults on ART with plasma HIV RNA below
40 copies/ml.
Methods: Seven HIV-positive adults received 12 weeks of ART intensification with
raltegravir alone or in combination with efavirenz or darunavir. Gut cells were obtained
by upper and lower endoscopy with biopsies from duodenum, ileum, colon, and rectum
at baseline and 12 weeks. Study outcomes included plasma HIV RNA, HIV DNA and
RNA from peripheral blood mononuclear cells (PBMC) and four gut sites, T-cell subsets,
and activation markers.
Results: Intensification produced no consistent decrease in HIV RNA in the plasma,
PBMC, duodenum, colon, or rectum. However, five of seven participants had a
decrease in unspliced HIV RNA per 10
6
CD4
þ
T cells in the ileum. There was a trend
towards decreased T-cell activation in all sites, which was greatest for CD8
þ
T cells in
the ileum and PBMC, and a trend towards increased CD4
þ
T cells in the ileum.
Conclusion: Most HIV RNA and DNA in the blood and gut is not the result of ongoing
replication that can be impacted by short-term intensification with raltegravir. How-
ever, the ileum may support ongoing productive infection in some patients on ART,
even if the contribution to plasma RNA is not discernible.
ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
AIDS 2010, 24:24512460
Keywords: ART, gut, HIV, intensification, intestine, raltegravir, T cell
Introduction
Although combined antiretroviral therapy (ART) can
reduce plasma viral load below 40 copies/ml, HIV persists
as latent provirus, low-level plasma RNA, and cell-
associated HIV RNA in peripheral blood mononuclear
cells (PBMCs) and lymphoid tissues. It is unclear whether
this residual HIV RNA results solely from reactivation of
a
San Francisco VA Medical Center (SFVAMC) and University of California, San Francisco (UCSF), San Francisco, California, USA,
b
University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology, University of Zurich, Zurich, Switzerland,
c
San Francisco General Hospital and University of California, San Francisco (UCSF), and
d
Department of Public Health, San
Francisco, California, USA.
Correspondence to Steven Yukl, 4150 Clement Street (111W3), San Francisco, CA 94121, USA.
Tel: +1 415 624 9088; fax: +1 415 379 5600; e-mail: steven.yukl@ucsf.edu
Received: 19 March 2010; revised: 25 June 2010; accepted: 2 August 2010.
DOI:10.1097/QAD.0b013e32833ef7bb
ISSN 0269-9370 Q 2010 Wolters Kluwer Health | Lippincott Williams & Wilki ns
2451
Page 1
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
latent infection [19] or whether there is a contribution
from cryptic, low-level but continuous rounds of new
infection [1019] or chronically-productive cells.
Because persistent replication or chronic infection would
require very different therapeutic strategies from those
aimed at latent infection, the question is critically
important for strategies aimed at eradication.
One approach to assess for ongoing replication is by
intensification of suppressive ART with one or more
additional agents whose activity affects a distinct part of
the viral life cycle. Several recent studies of ART
intensification in patients on contemporary ART with
viral load below 4050 copies/ml failed to show a
reduction in plasma HIV RNA [1,5,8,9,19]. However,
the plasma may not reflect changes elsewhere in the body,
especially the lymphoid tissues, which are the major sites
of HIV infection in untreated patients. Given the role of
the gut-mucosal lymphoid compartment as a reservoir for
HIV, we hypothesized that intensification may result in
reductions in HIV RNA and/or immune activation that
may be detected in the gut. Because different regions of
the gut may vary in their ability to support productive
infection, we performed a pilot prospective study to
examine the effect of ART intensification with raltegravir
(RLG) alone or in combination with a second agent on
viral burden and T-cell activation in multiple regions of
the gut.
Methods
Patient recruitment
HIV-positive adults meeting entry criteria were recruited
from two hospital-based HIV clinics. Inclusion criteria
included: age 1865 years; infection with HIV-1; ART
for at least 12 months; no change in ART for at least
3 months; CD4
þ
T-cell count above 200 cells/ml; and
viral load below 40 copies/ml for more than 6 months
prior to study entry. Exclusion criteria included factors
that would increase the risk from sedation, endoscopy,
biopsy, or intensification. The study was approved by
the Institutional Review Board of the University of
California, San Francisco.
Study design
The study was an open-label, pilot interventional study of
16 weeks duration. All participants had baseline clinical
and laboratory evaluations followed by esophagogastro-
duodenoscopy (EGD) with 710 biopsies from the
duodenum and colonoscopy with 710 biopsies each
from the terminal ileum, right colon, and rectum. After
baseline endoscopy, the ART regimen was intensified by
adding RLG with or without a study protease inhibitor or
non-nucleoside reverse transcriptase inhibitor (NNRTI)
for 12 weeks. Participants who were not already on an
NNRTI and who were suitable candidates had the option
of adding a study NNRTI, whereas those who were not
on a protease inhibitor had the option of adding a study
protease inhibitor. Additional laboratory and clinical
evaluations were done at weeks 1, 4, and 12. Repeat EGD
and colonoscopy with biopsies were performed at week
12. Intensification was then stopped, and a final blood
draw was done at week 16.
Endoscopy and biopsy
Esophagogastroduodenoscopy, colonoscopy, and biopsies
were performed as described previously (S. Yukl, S.
Gianella, E. Sinclair, et al., in preparation).
Processing of gut biopsies
Gut biopsies were immediately placed in Roswell
Park Memorial Institute (RPMI) medium with L-Glu,
penicillin/streptomycin, and 15% fetal calf serum for
subsequent cell isolation. Six to nine biopsies from each
site were separated into single cells using a modification of
a published method incorporating both collagenase
digestion and needle shearing (S. Yukl, S. Gianella, E.
Sinclair, et al., in preparation) [20,21]. Briefly, biopsies
were subjected to three rounds of collagenase digestion,
mechanical disruption (by passing through a blunt 16-
gauge needle), clarification (by passing through a 70 mm
cell strainer), and washing. The three aliquots of strained
and washed gut cells were then combined, counted, and
resuspended in phosphate-buffered saline (PBS) þ 0.1%
bovine serum albumin (BSA) þ2 mmol/l ethylene
diamine tetraacetic acid (EDTA). 5 10
5
cells were set
aside for flow cytometry, whereas the remainder was
divided and frozen at 808C for subsequent DNA or
RNA extraction. Of note, previous studies have docu-
mented that HIV DNA and RNA can be reproducibly
quantified from a single endoscopic biopsy [22,23].
Processing of blood
Sixty-eight millilitres of blood was collected for plasma
and PBMC in Vacutainer ACD tubes with Solution A
(BD). Plasma was obtained by centrifuging twice at 1000g
for 10 min without braking. The buffy coat from the first
spin was used to isolate PBMC by centrifugation on Ficoll
according to the procedure recommended for Dynal
Invitrogen Bead Separations. PBMCs were resuspended
in PBS þ 0.1% BSA þ 2 mmol/l EDTA. Aliquots of
PBMCs were collected for flow cytometry, HIV DNA,
and HIV RNA. 6 10
7
PBMCs were used to isolate
CD4
þ
cells by negative selection using the Dynabeads
Untouched Human CD4
þ
T Cell Kit (Invitrogen).
5 10
5
CD4
þ
T cells were saved for flow cytometry,
whereas the remainder was frozen as cell pellets for HIV
DNA and RNA.
Immunophenotyping
Antibodies and fluorochromes included the following:
CD45-APC, CD3-Pacific Blue (BD Bioscience), CD4-
ECD (Beckman Coulter), CD8-Q-Dot605 (Invitrogen,
Custom conjugate), CD38-PE (BD Bioscience), and
2452 AIDS 2010, Vol 24 No 16
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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
HLA-DR-FITC (BD Bioscience). Staining, washing,
fixing, flow cytometry, and data analysis were performed
as described previously (S. Yukl, S. Gianella, E. Sinclair,
et al., in preparation) [24]. Cells were gated on a scatter
plot to remove debris, then sequentially gated for CD45
(to define total leukocytes), CD3 (to define T cells), and
CD4 or CD8. CD38 and HLA-DR gates were set using
Fluorescent-Minus-One controls for each marker on a
PBMC sample then applied to PBMC and gut samples
from the same participant. For each participant, gating
was consistent between weeks 0 and 12.
High-volume plasma HIV RNA
Plasma HIV RNA was measured using a modification of
the Abbott m2000 assay [25,26] with limit of detection
(LOD) of less than 0.5 copies/ml (S. Yukl, P. Li, K.
Fujimoto, et al., in preparation). Plasma (30 ml) was diluted
1 : 1 with PBS, divided in two portions, layered onto 10 ml
of 6% iodixanol [OptiPrep Density Gradient Medium
(Sigma) diluted 1 : 10 in PBS] in 50 ml polypropylene tubes
(Beckton), and centrifuged at 47 810g for 3 h at 48C
without braking. Viral pellets were resuspended in a total of
1000 ml of PBS and the HIV RNA was measured accord-
ing to the Abbott M2000 protocol. Copy values were
extrapolated from the Ct values of the standards and then
adjusted for the concentration factor.
Cell-associated HIV DNA by real-time PCR
DNA was extracted using the DNA Blood Mini Kit
(Qiagen) and measured using a NanoDrop 1000 Spectro-
photometer. Three replicates of 500 ng of DNA from each
sample were then used in a real time Taqman PCR assay for
HIV DNA that uses primers and a Locked Nucleic Acid
probe from the Gag region. This assay detected a 10 copy
standard 19/20 times (95%) with a mean value of 9.5 þ/
1.7 copies. External standards (10
5
to 1) were prepared
from DNA extracted from serial dilutions of known
numbers of 8E5 cells (NIH AIDS Reagent Program), each
of which contains one integrated HIV genome per cell.
HIV DNA copy numbers were extrapolated from the Ct
values of the samples and expressed as copies/10
6
cells
(assuming 1 mg total DNA corresponds to 160 000 cells).
To account for variation in the number of CD4
þ
T cells
in different samples, results were also normalized by the
percentage of all cells that were CD45
þ
CD3
þ
CD4
þ
(by
flow cytometry) and expressed as copies/10
6
CD4
þ
T cells. To verify the DNA concentrations and assess for
PCR inhibitors, samples from four patients were assayed
using a separate real time PCR for b-actin.
Cell-associated HIV RNA by reverse
transcriptase real-time PCR (qRT-PCR)
RNA was extracted using the Rneasy Kit (Qiagen) with
on-column digestion using RNase-free DNase (Qiagen).
To maximize the sensitivity of the qRT-PCR assays,
which approaches one copy per reaction [27,28], primers
and probes were matched to each participant based on the
sequence of the prevailing viral quasispecies in peripheral
CD4
þ
T-cell DNA. Unspliced HIV RNA (UsRNA) was
measured using primers from the pol region (25362562
and 26342662). Multiply-spliced HIV RNA encoding
for Tat and Rev (MsRNA
tat-rev
) was measured using
primers from tat exon 1 (59565979) and tat/rev exon
2 (84338459). Total multiply-spliced HIV RNA
(MsRNA
To t
) was measured using primers from rev exon
1 (60126045) and tat/rev exon 2 (84338459). Probes
were chosen from a panel of highly conserved wild-type
probes that matched their target sequences by 100% [29],
except for MsRNA from patient 196, for which no
matching wild-type probe could be identified, and for
which an individualized probe was designed. qRT-PCR
was done under the conditions described previously [30].
HIV-1 RNA copy numbers (the mean of duplicate PCR
measurements) were calculated as described previously (S.
Yukl, S. Gianella, E. Sinclair, et al., in preparation) [27] and
then normalized to the cellular input into the PCR, as
determined both by total RNA concentration (measured
by NanoDrop 1000, assuming that 1 ng RNA correspond
to 1000 cells [31]) and by levels of glyceraldehyde
phosphate dehydrogenase (GAPDH) RNA (as determined
by a separate qRT-PCR). Results (copies/10
6
cells) from
the two different methods of normalization correlated
well. To account for variation in the number of CD4
þ
T cells in different samples, HIV RNA copy numbers were
also normalized by the percentage of all cells that were
CD45
þ
CD3
þ
CD4
þ
(by flow cytometry).
Statistics
Results from week 0 and week 12 were compared across
all participants using the Wilcoxon signed rank test.
Results
Study population
Of 14 patients who were screened, 13 met study criteria
and 8 consented to enter the study. The eight participants
had a median age of 51 years, median duration of HIV
infection of 14.5 years, and median entry CD4 of
473 cells/ml (Table 1). They had maintained viral load
below 40 copies/ml for 2.812 years and had a median
baseline viral load of 2.3 copies/ml. Five participants were
intensified with RLG alone; the remainder added a
protease inhibitor or NNRTI in addition to RLG. One
participant (A190) withdrew shortly after study entry for
personal reasons. There were no serious adverse events
attributed to intensification or endoscopic biopsies.
Plasma HIV RNA
Plasma HIV RNA was undetectable in all study parti-
cipants using the standard Abbott assay, but was detectable
in all participants using the high-volume HIV RNA assay
with LOD of less than 0.5 copy/ml (S. Yukl, S. Gianella,
Effect of RLG intensification on HIV in the gut Yukl et al. 2453
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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
E. Sinclair, et al., in preparation). Mean plasma viral loads
ranged from 0.5 to 6 copies/ml, with a median of
2.3 copies/ml (Table 1). Intensification resulted in no
consistent decrease in plasma HIV RNA (Fig. 1a).
Cell-associated HIV RNA and DNA in peripheral
blood
At baseline, unspliced HIV RNA was detectable in the
blood of all participants, with a median of 44 copies/10
6
cells in PBMC and 280 copies/10
6
cells in peripheral
CD4
þ
T cells (S. Yukl, S. Gianella, E. Sinclair, et al., in
preparation). There was no consistent change in Us HIV
RNA in PBMC (Fig. 1b) or peripheral CD4
þ
T cells (data
not shown) during or following intensification. Cell-
associated HIV DNA was detectable in the blood of all
participants at baseline, with a median of 591 copies/10
6
cells in PBMCs and 6902 copies/10
6
cells in peripheral
CD4
þ
T cells. Two participants, A185 and A186, appeared
to have a decrease in HIV DNA in both PBMC (Fig. 1c)
and peripheral CD4
þ
T cells (Fig. 1d) during the period
of intensification. In both participants, the decrease in
HIV DNA in PBMCs was sustained after cessation of
intensification (week 16), whereas the decrease in HIV
DNA in CD4
þ
T cells was almost completely reversed by
week 16.
Cell-associated HIV RNA and DNA in gut
At baseline, unspliced but not multiply-spliced HIV RNA
could be detected in the majority of participants at each gut
site (S. Yukl, S. Gianella, E. Sinclair, et al., in preparation).
Since the yield of CD4
þ
T cells varied between sites and
time points, levels of unspliced HIV RNA/10
6
cells were
normalized to CD4
þ
T-cell content using the results from
flow cytometry. From week 0 to week 12, five of seven
participants had a decrease in unspliced RNA/10
6
CD4
þ
T cells in the terminal ileum (Fig. 2b). For the seven
participants, the mean unspliced HIV RNA/10
6
CD4
þ
T-
cells in the ileum decreased from 3438 to 682 copies/10
6
CD4
þ
T-cells (Fig. 2a; mean change: 2520 copies/10
6
CD4
þ
T-cells; median change: 576). There was no
consistent change in unspliced RNA in peripheral CD4
þ
T-cells (mean change: 100 copies/10
6
CD4
þ
T-cells),
duodenum (mean change: 86), colon (mean change: 393),
or rectum (mean change: 145).
At baseline, HIV DNA was detectable in each gut site in
the majority of patients (S. Yukl, S. Gianella, E. Sinclair,
et al., in preparation). Intensification resulted in no
consistent decrease in HIV DNA/10
6
CD4
þ
T-cells in
the PBMC (mean change: 344 copies/10
6
CD4
þ
T cells),
ileum (mean change: 6580), colon (mean change:
1300), or rectum (mean change: 13260), though
all participants had an increase in HIV DNA/10
6
CD4
þ
T-cells in the duodenum (Fig. 2c).
CD4 cell count and percentage
As measured by the clinical lab, five of seven participants
had an increase in the peripheral CD4% (from mean of
2454 AIDS 2010, Vol 24 No 16
Table 1. Clinical characteristics.
Individual Age Sex
Years
HIV
Total time
on ART
(years)
Total duration
VL <40
(years)
Time since
last VL >40
(years)
Mean entry
VL (copy/ml)
CD4 nadir
(#/ul)
Entry CD4
(#/ul/%) Entry regimen Intensification
A185 60 M 14 3.8 2.8 2.8 6.0 49 348/21% ABC/3TC/ATV RAL
A186 44 M 12 8.0 3.2 3.2 5.4 400 894/35% FTC/TDF/EFV RAL/DRV/r
A188 55 M 22 12.0 12.0 12.0 0.9 98 289/20% FTC/TDF/ATV/r RAL/EFV
A189 55 M 15 5.2 4.9 2.2 3.3 51 365/20% FTC/TDF/ATV/r RAL/EFV
A190
a
33 M 11 10.3 8.4 6.9 0.5 469 1552/53% FTC/TDF/EFV RAL
A195 63 M 21 10.8 9.8 8.3 0.5 188 437/32% TDF/ABC/NVP/LPV/r RAL
A196 33 M 14 11.0 10.5 4.3 2.1 350 751/36% FTC/TDF/EFV RAL
A198 47 M 22 12.7 4.9 2.8 2.4 250 518/42% FTC/TDF/NVP/ATV/r/MVC RAL
Median 51 N/A 14.5 10.5 6.7 3.7 2.3 219 478/34%
3TC, lamivudine; ABC, abacavir; ATV, atazanavir; DRV, darunavir; EFV, efavirenz; FTC, emtricitabine; LPV, lopinavir; MVC, maraviroc; N/A, not applicable; NVP, nevirapine; r, ritonavir; RAL,
raltegravir; TDF, tenofovir; VL, viral load.
a
A190 withdrew consent shortly after study entry.
Page 4
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
29.43 to 30.57%; median change þ1%) from week 0 to
12, though there was no consistent change in absolute
CD4 cell counts. Relative numbers of CD4
þ
T cells (as a
percentage of all cells and percentage of CD3
þ
T cells)
were also measured in isolated PBMC and suspensions of
gut cells using flow cytometry. Six of seven participants
had an increase in CD4
þ
T cells (as percentage of all) in
the terminal ileum (from mean of 1.93 to 2.85%; median
change þ0.49%) and in the right colon (from mean of
2.55 to 3.85%; median change þ1.13%) but not other
sites (Fig. 3a). Five of seven participants had an increase in
CD4% in the ileum (from mean of 27.46 to 33.52%;
median change þ9.88%), whereas changes in other sites
were less consistent (Fig. 3b).
Activation of CD4
R
and CD8
R
T cells
Intensification resulted in a trend towards decreased
activation (CD38
þ
, HLA-DR
þ
, and dualþ) of CD4
þ
and CD8
þ
T cells in all sites, which was greatest for
CD38
þ
and for CD8
þ
T cells in the PBMC, ileum, and
colon. Six of seven participants had a decrease in CD38
þ
T cells as percentage of CD8
þ
T cells (Fig. 4b) in PBMC
(from mean of 40.61 to 36.43%; median change 4.84%)
and colon (from mean of 89.90 to 87.90%; median
change 1.11%). Five and six (respectively) out of seven
also had a decrease in CD38
þ
HLA
DR
þ
cells as
percentage of CD8
þ
T cells (Fig. 4f) in the PBMC
(from 15.24 to 13.21%; median change 1.9%) and colon
(from 46.94 to 42.63%; median change 4.27%). For
both CD4
þ
and CD8
þ
T cells, the mean decreases in
percentage of HLA
DR
þ
and CD38
þ
HLA
DR
þ
T cells
were greatest in the terminal ileum.
Discussion
The effect of RLG-containing ART intensification on
viral burden, T-cell reconstitution, and T-cell activation
was investigated in seven chronically-infected HIV-
positive men who had viral load below 40 copies/ml
for a median of 6.7 years. Despite the limitations of study
size and sampling, this study provided several pertinent
findings. First, we confirmed the observation by others
[1,5,8,9,19] that ART intensification does not reduce
HIV RNA in the plasma, even though we intensified
with one or two new agents and applied an assay with
LOD of less than 0.5 copy/ml. This finding, coupled with
sequencing studies that show no evidence of drug
resistance and little evolution of the plasma RNA over
time [24], suggests that low-level plasma virus is not due
to ongoing viral replication in circulating lymphocytes.
Effect of RLG intensification on HIV in the gut Yukl et al. 2455
15
10
5
0
50 5101520
Week
Plasma HIV RNA vs. time
(a) (b)
(c) (d)
HIV DNA in PBMC vs. time HIV DNA in peripheral CD4
+
T cells vs. time
Unspliced HIV RNA in PBMC vs. time
50 5101520
Week
505101520
Week
50 5101520
Week
Plasma HIV RNA
(copies/ml)
0
1000
2000
3000
HIV DNA (copy/million cells)
0
5000
10000
HIV DNA (copy/million cells)
0
100
200
A185
A186
A188
A189
A195
A196
A198
A185
A186
A188
A189
A195
A196
A198
A185
A186
A188
A189
A195
A196
A198
A185
A186
A188
A189
A195
A196
A198
300
Us HIV RNA
(copies/million cells)
Fig. 1. Blood HIV levels as a function of time through week 16. (a) Shows the plasma HIV RNA, as measured by the high-volume
plasma HIV RNA assay. (b) Shows the cell-associated unspliced HIV RNA in peripheral blood mononuclear cells (PBMCs), as
measured by real-time reverse transcriptase PCR. (c, d) Show the cell-associated HIV DNA in PBMC (c) and sorted peripheral
CD4
þ
T cells (d), as measured by real-time PCR. The X-axis shows the study week. Intensification was started after week 0 and
stopped at week 12.
Page 5
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Second, we extended these findings by showing that
intensification results in no consistent change in cell-
associated HIV RNA in either blood (PBMC or CD4
þ
T cells) or in the duodenum, colon, or rectum. This
finding suggests that most of the HIV RNA in the blood
and many regions of gut is not the result of ongoing
replication that can be impacted by short-term intensi-
fication with RLG-containing regimens. However, in the
ileum, which has the highest baseline HIV unspliced
RNA and RNA/DNA ratio, the unspliced RNA/10
6
CD4
þ
T-cells decreased in five of seven participants in
conjunction with intensification. Supporting the inter-
pretation that intensification affected viral production, we
also observed a trend towards an increase in CD4 content
in the ileum and a decrease in CD8
þ
T-cell activation in
the ileum and blood. Whereas several recent studies argue
against productive infection in the blood of ART-
suppressed patients, other findings in ART-suppressed
patients would be consistent with ongoing replication.
These findings include the presence of 2-LTR circles and
2456 AIDS 2010, Vol 24 No 16
Fig. 2. Change in cell-associated unspliced HIV RNA (a, b)
and HIV DNA (c) per 10
6
CD4
R
T cells. HIV copy numbers
were measured by real-time PCR, normalized for the total cell
input into the PCR (by mg RNA or DNA), and then normalized
to the percentage of all cells that were CD4
þ
T cells (by flow
cytometry). (b) Shows the HIV RNA values in the ileum for
each participant at weeks 0 and 12. In (a) and (c), column
heights indicate the average of the changes (week 12week 0)
in HIV copy number per 10
6
CD4
þ
T cells, as measured from
peripheral blood mononuclear cells or total gut cells
(obtained by collagenase digestion of endoscopic biopsies)
from each of the four gut sites. Error bars indicate the standard
error of measurement (SEM).
Fig. 3. Change in CD4
R
T cells as a percentage of all cells (a)
and as a percentage of T cells (b) by site. The Y-axis shows the
average difference between the percentage of CD4
þ
T cells at
week 12 and the percentage at week 0, as measured by flow
cytometry from peripheral blood mononuclear cells (PBMCs)
or total gut cells (obtained by collagenase digestion of endo-
scopic biopsies) from each of the four gut sites. Error bars
indicate the SEM.
Page 6
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
functional unintegrated HIV DNA [10,11,19], the fact
that there is more HIV DNA in activated CD4
þ
T cells
than resting CD4
þ
T cells [12], and the fact that immune
activation remains high despite years of ART [13].
Moreover, ART intensification reduces the half-life of
the latent reservoir [14,17], reduces plasma RNA [15],
increases CD4
þ
cell counts [15,16], and reduces immune
activation [1519].
Studies by ourselves and others have shown that HIV
DNA per CD4
þ
T cell is disproportionately concentrated
(up to 10-fold higher) in the gut relative to peripheral
blood [32], which has been attributed to the presence of
cryptic low-level ongoing replication in the gut [32]. If
the much higher concentration of HIV DNA in gut
CD4
þ
T cells reflects substantial numbers of newly
infected cells, one would expect a significant proportion
of the HIV DNA to be in the labile unintegrated form. If
intensification resulted in more complete suppression,
one would expect that these labile forms would be cleared
[33]. However, we found that intensification resulted in
no consistent decrease in HIV DNA in the gut. The lack
of change in gut HIV DNA with intensification suggests
that most of this DNA represents latently infected cells,
chronically infected cells, or defective provirus rather than
the result of new infectious events that can be prevented
by RLG.
In two participants (A185 and A186), there was a decrease
in HIV DNA in both the PBMC and the peripheral
CD4
þ
T cells, which was reversed at week 16 in the
CD4
þ
T cells but not in the PBMC. This finding suggests
that HIV DNA may have a different nature, origin, or
stability in different cell types within the blood. Of note,
Effect of RLG intensification on HIV in the gut Yukl et al. 2457
Fig. 4. Change in T-cell activation by site. Using flow cytometry, T-cell activation was measured by the percentage of CD4
þ
T cells (left column) or CD8
þ
T cells (right column) that express both CD38 and HLA-DR (a, b) as well as the percentage of the total
that express CD38 (c, d) and the percentage of the total that express HLA-DR (e, f). The Y-axis shows the average difference
between the percentage of activated T cells at week 12 and the percentage at week 0, as measured from peripheral blood
mononuclear cells (PBMCs) or total gut cells (obtained by collagenase digestion of endoscopic biopsies) from the indicated gut
sites. Error bars indicate the SEM.
Page 7
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
this proportion is similar to the proportion of patients
undergoing RLG intensification who experienced
transient rises in the concentration of 2-LTR circles, a
finding which was attributed to recent infections aborted
by RLG [19].
Findings from this study raise new questions. If there is
ongoing replication in the ileum, then why is a reduction
in RNA not observed in the plasma? The simplest
explanation is that the ileum contributes only a small
fraction of the total HIV RNA that is measurable in the
plasma, perhaps because there is relative compartmenta-
lization (or delayed equilibration) between the two
compartments or infection is aborted after local replica-
tion in the ileum. Other mechanisms, such as reactivation
of latently-infected cells, release of virions captured on
the follicular dendritic network, or chronic transcrip-
tionally-active cells, could be responsible for the majority
of residual HIV RNA in the blood. It is also unclear why
ART intensification had differential effects in the four gut
sites. Gut sites could differ in penetration of ART agents,
immunologic environments, or the composition of
CD4
þ
T-cell populations. The ileum, which is an
immune inductive site and is enriched for lymphoid
aggregates, seems disproportionately involved in other
inflammatory processes such as Crohn’s disease and
enteric infection with tuberculosis, Salmonella, and
Yersinia. Close proximity of CD4
þ
T cells in the lym-
phoid aggregates may create an environment that is more
conducive to cell-to-cell spread of HIV [3443].
However, one must question whether low-level ongoing
replication in the ileum has true consequences for HIV
persistence and eradication strategies.
Limitations of our study must be acknowledged. First, we
had a relatively small number of participants and were
unable to sample extensively at all four gut sites. However,
six to nine biopsies from each site were used to isolate gut
cells, and the relative and absolute levels of HIV and
immune markers were remarkably consistent from week 0
to 12. Second, there was some heterogeneity in baseline
clinical characteristics, entry regimens, and intensification
regimens, which may account for some of the differences
in responses to intensification. Third, the period of
intensification may have been too short relative to the
clearance rate of virions or newly infected cells. Fourth,
inflammation and microbial leakage from the biopsies
could have confounded the results and could perhaps
account for the observed increase in HIV DNA in the
duodenum. Finally, all studies aimed to detect low-level
replication suffer from the difficulty that such infectious
events could occur in minute, temporally and spatially
discrete foci [44].
Despite these limitations, the results add to our under-
standing of the multiple mechanisms that contribute to
the persistence of HIV in patients on ART. The lack of
significant intensification-mediated changes in viral
RNA in blood and most gut sites implies a process
other than the classic model of ongoing replication, such
as reactivation from latency or chronic persistence of
virions and/or chronically infected, transcriptionally
active cells. At the same time, the changes in immune
activation and CD4 cell counts would be consistent with
ongoing replication in some patients, either in the ileum
or at another site. Future studies are needed to confirm
the presence and site(s) of ongoing replication and to
identify clinical and virological factors that may predict
the contribution of ongoing replication to viral persis-
tence. Whereas some patients may potentially derive
benefit from intensification of ART, other strategies will
be necessary for viral eradication.
Acknowledgements
We thank the following: the study participants; PLUS staff
members Michele Downing and Marc Gould; VA study
nurses Sandra Charles and Linda Adams; members of the
Wong lab (including Alex L. M. Choi, Peilin Li, and
Katsuya Fujimoto), the SFVAMC GI Endoscopy Unit,
the UCSF Core Immunology Lab (including Lorrie
Epling, Valerie Girling, and Terence Ho), and the San
Francisco Department of Public Health; and the NIH
AIDS Research Reagent Program. This work was
supported in part by the U.S. Department of Veterans
Affairs [VA Merit Review (J.W./S.A.Y.)], the National
Institute of Health [NIH grants P30-AI027763 (S.A.Y.),
NS051145 (J.W./S.A.Y.) and T32 AI60530 (D.H./
S.A.Y.)], and the Swiss National Science Foundation
[3100AO-112670 (M.F./H.F.G.) and 324730-130865
(H.F.G.)].
S.A.Y., A.K.S., K.M., H.L., C.B.H., E.S., H.F.G., M.F.,
J.K.W., and D.V.H. contributed to the study design.
A.K.S. and K.M. performed the endoscopies. S.A.Y. and
S.G. performed the laboratory work. S.A.Y., S.G., M.P.,
E.S., H.F.G., M.F., J.K.W., and D.V.H. contributed to
analysis and interpretation of data. S.A.Y., S.G., J.K.W.,
and D.V.H. wrote the manuscript. All authors con-
tributed to critical review of the manuscript.
Disclaimers: C.B.H. is an advisor to Merck and is on
their speakers’ bureau. No other author has a
commercial or other association that may pose a
conflict of interest. All research was conducted within
the guidelines of ethical principles, local legislation,
and the local institutional review board (IRB).
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2460 AIDS 2010, Vol 24 No 16
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  • Source
    • "Beyond the importance of identifying persistent virus in this anatomical location, a key finding in these works was the observation that virus coding for cytotoxic T lymphocytes (CTL) escape mutations were archived in the central nervous system during ART [22]. Surprisingly, given the intense focus on the intestines as a major anatomical site harboring persistent virus [20, 21, 29, 35,111112113114115116117118119120121122123124, there is no published evidence from humans or animal models that replication competent, latent HIV proviruses persist in the intestines during ART. Common to these anatomical location analyses were their reliance upon nucleic acid measures of viral persistence . "
    [Show abstract] [Hide abstract] ABSTRACT: Research challenges associated with understanding HIV persistence during antiretroviral therapy can be categorized as temporal, spatial and combinatorial. Temporal research challenges relate to the timing of events during establishment and maintenance of HIV persistence. Spatial research challenges regard the anatomical locations and cell subsets that harbor persistent HIV. Combinatorial research challenges pertain to the order of administration, timing of administration and specific combinations of compounds to be administered during HIV eradication therapy. Overcoming these challenges will improve our understanding of HIV persistence and move the field closer to achieving eradication of persistent HIV. Given that humanized mice and non-human primate HIV models permit rigorous control of experimental conditions, these models have been used extensively as in vivo research platforms for directly addressing these research challenges. The aim of this manuscript is to provide a comprehensive review of these recent translational advances made in animal models of HIV persistence.
    Full-text · Article · Dec 2016 · Journal of Translational Medicine
  • Source
    • "Regarding the results on rectal proviral DNA, we cannot conclude due to the small sample size and no control group for this test. Nevertheless, previous studies have shown that rectal biopsies might not be the best representative of gut HIV reservoirs, compared to other locations like the ileum [18]. One other possible confounding factor is the fact that we included HIV naïve patients with a short time of undetectable viremia on cART. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Ongoing HIV-1 replication in lymphoid cells is one explanation of the persistence of HIV-1 reservoirs despite highly active antiretroviral therapy (cART). We tested the potential of cART intensification by Maraviroc plus Raltegravir to decrease proviral HIV-1 DNA levels in lymphoid cells during a randomized trial. Patients and methods: We randomly assigned for 48 weeks 22 patients to continue their current first line regimen of Truvada (R) plus Kaletra (R) or intensify it with Maraviroc and Raltegravir. The primary objective was to obtain a 50% decrease in proviral HIV-1 DNA levels in lymphoid cells with intensification. Blood samples were drawn at W-2, W0, W2, W4, W12, W24 and W48. Plasma viremia, cellular proviral DNA and cellular RNA, 2-LTR circles and lymphocytes subsets were assayed using validated methods. Patients in the intensified group underwent a gut biopsy at baseline and W48 to measure proviral DNA levels. Statistical analysis used parametric and non-parametric tests. Results: Ten patients in each arm completed the trial. The 2 populations were comparable at baseline. No change in the reservoir size was observed in the intensified arm compared to the control arm measured in peripheral blood mononuclear cells (PBMCs). No change in the reservoir size was observed in gut proviral DNA in the intensified arm. In this group, no increase in 2-LTR circles was observed as early as 2 weeks after intensification and no change was found in residual plasma RNA levels measured by the single copy assay. However, a decrease in CD8(+) T cells activation was observed at 24 and 48 weeks, as well as in PBMCs HIV-1 RNA levels. Conclusion: We conclude that the intensification of a Protease Inhibitor regimen with Maraviroc and Raltegravir does not impact the blood proviral DNA reservoir of HIV but can decrease the cell-associated HIV RNA, the CD8 activation and has a possible impact on rectal proviral HIV DNA in some patients.
    Full-text · Article · Oct 2014 · AIDS Research and Therapy
  • Source
    • "However, the source of this residual viremia remains controversial, with competing arguments supporting its origin from incompletely suppressed viral replication (especially in tissues), vs. suggestions that it arises from stochastic reactivation of latently infected cells, or from persistently infected cells not susceptible to inhibition by cART, and/or resistant to immune clearance. Numerous cART intensification studies have attempted to address this question, with most showing no impact, although a couple of studies have demonstrated apparent effects of treatment intensification, particularly when adding integrase inhibitors to base regimens including protease inhibitors [28], [29] [30]. "
    [Show abstract] [Hide abstract] ABSTRACT: Objectives Viral reservoirs–persistent residual virus despite combination antiretroviral therapy (cART)–remain an obstacle to cure of HIV-1 infection. Difficulty studying reservoirs in patients underscores the need for animal models that mimics HIV infected humans on cART. We studied SIV-infected Chinese-origin rhesus macaques (Ch-RM) treated with intensive combination antiretroviral therapy (cART) and 3 weeks of treatment with the histone deacetyalse inhibitor, suberoylanilide hydroxamic acid (SAHA). Methods SIVmac251 infected Ch-RM received reverse transcriptase inhibitors PMPA and FTC and integrase inhibitor L-870812 beginning 7 weeks post infection. Integrase inhibitor L-900564 and boosted protease inhibitor treatment with Darunavir and Ritonavir were added later. cART was continued for 45 weeks, with daily SAHA administered for the last 3 weeks, followed by euthanasia/necropsy. Plasma viral RNA and cell/tissue-associated SIV gag RNA and DNA were quantified by qRT-PCR/qPCR, with flow cytometry monitoring changes in immune cell populations. Results Upon cART initiation, plasma viremia declined, remaining <30 SIV RNA copy Eq/ml during cART, with occasional blips. Decreased viral replication was associated with decreased immune activation and partial restoration of intestinal CD4+ T cells. SAHA was well tolerated but did not result in demonstrable treatment-associated changes in plasma or cell associated viral parameters. Conclusions The ability to achieve and sustain virological suppression makes cART-suppressed, SIV-infected Ch-RM a potentially useful model to evaluate interventions targeting residual virus. However, despite intensive cART over one year, persistent viral DNA and RNA remained in tissues of all three animals. While well tolerated, three weeks of SAHA treatment did not demonstrably impact viral RNA levels in plasma or tissues; perhaps reflecting dosing, sampling and assay limitations.
    Full-text · Article · Jul 2014 · PLoS ONE
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