Whole-body immunoPet reveals active siV dynamics in viremic and

Transcription

Articles
Whole-body immunoPET reveals active SIV dynamics
in viremic and antiretroviral therapy–treated
macaques
© 2015 Nature America, Inc. All rights reserved.
Philip J Santangelo1, Kenneth A Rogers2, Chiara Zurla1, Emmeline L Blanchard1, Sanjeev Gumber3,4, Karen Strait5,
Fawn Connor-Stroud5, David M Schuster6, Praveen K Amancha2, Jung Joo Hong2, Siddappa N Byrareddy4,
James A Hoxie7, Brani Vidakovic1, Aftab A Ansari4, Eric Hunter4 & Francois Villinger2,4
The detection of viral dynamics and localization in the
context of controlled HIV infection remains a challenge and
is limited to blood and biopsies. We developed a method
to capture total-body simian immunodeficiency virus (SIV)
replication using immunoPET (antibody-targeted positron
emission tomography). The administration of a poly(ethylene
glycol)-modified, 64Cu-labeled SIV Gp120–specific antibody
led to readily detectable signals in the gastrointestinal and
respiratory tract, lymphoid tissues and reproductive organs
of viremic monkeys. Viral signals were reduced in aviremic
antiretroviral-treated monkeys but detectable in colon, select
lymph nodes, small bowel, nasal turbinates, the genital tract
and lung. In elite controllers, virus was detected primarily in
foci in the small bowel, select lymphoid areas and the male
reproductive tract, as confirmed by quantitative reversetranscription PCR (qRT-PCR) and immunohistochemistry. This
real-time, in vivo viral imaging method has broad applications
to the study of immunodeficiency virus pathogenesis, drug and
vaccine development, and the potential for clinical translation.
that can possibly serve as viral reservoirs so that the mechanisms
by which such reservoirs are maintained can be identified. This
would facilitate the development of strategies for eliminating
these reservoirs, particularly in individuals treated with highly
active antiretroviral therapy (ART)5. Ideally, a method to identify
changes in virus localization would be minimally invasive as
well as specific, sensitive and amenable to repeated application.
Here we describe the application of whole-body imaging to the
detection and localization of sites of SIV infection in chronically
infected, ART-treated and EC macaques.
Delineating viral replication in the context of generalized
infections has traditionally relied on indirect measures, such as
evaluating viral loads in plasma or via specific tissue biopsies.
Such approaches have been valuable for the clinical management
of viral infections, although they generally do not identify the
site or source of virus replication in vivo. In a small percentage
of HIV-infected individuals termed elite controllers (ECs), virus
replication is controlled to undetectable levels without anti
retroviral intervention, and disease progression may be delayed
for decades1,2. Despite undetectable virus in the plasma, virus
evolution continues to occur consistently with ongoing tissuecontained virus replication3,4. It is critical to identify tissue sites
Development of the immunoPET probe
We selected the SIV Env protein–specific monoclonal antibody (mAb) clone 7D3 as the basis of the probe because of
its broad SIV Env specificity13–15. 7D3 binds the CCR5 binding site of Gp120 and prevents syncytia formation in vitro with
SIVMACCP-MAC, although it does not affect soluble CD4 binding or
neutralize SIVmac239 (ref. 13). Furthermore, three 7D3 molecules
can bind to the trimeric Env of SIVMACCP-MAC and SIVMAC239
(ref. 15). To mitigate the immunogenicity of murine antibodies
(probe antibodies were not detected after two administrations;
data not shown), decrease nonspecific interactions and enable
the chelation of 64Cu, the mAb was modified with linear 10-kDa
RESULTS
We hypothesized that the higher sensitivity and image quality of
64Cu-based positron emission tomography (PET) over that of
single-photon emission computer tomography6 could be exploited
to detect the major target tissues infected by SIV or HIV by scanning the entire body7–10. To test this hypothesis, we employed the
SIV-infected rhesus macaque as a model of pathogenic HIV infection11,12 and chose the glycoprotein Gp120 as the in vivo target7.
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA. 2Division of Microbiology
and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USA. 3Division of Pathology, Yerkes National Primate Research
Center, Emory University, Atlanta, Georgia, USA. 4Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
5Division of Veterinary Medicine, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USA. 6Department of Radiology and Imaging Sciences,
Emory University School of Medicine, Atlanta, Georgia, USA. 7Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Correspondence should be addressed to F.V. ([email protected]).
Received 21 June 2014; accepted 7 February 2015; published online 9 march 2015; CORRECTED AFTER PRINT 11 JUNE 2015; doi:10.1038/nmeth.3320
nature methods | VOL.12 NO.5 | MAY 2015 | 427
Articles
Viral RNA
(copies per ng), by PCR
428 | VOL.12 NO.5 | MAY 2015 | nature methods
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ester–amino chemistry and the chelator DOTA NHS (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid mono), which
has been demonstrated to be stable in humans and mice for at
least 72 h (refs. 17,18).
We performed in vitro assays to validate the probe
(Supplementary Fig. 1). First, we incubated the 64Cu-labeled
radiotracer with varying percentages of SIV1C cells and uninfected Hut78 cells (Supplementary Fig. 1a), demonstrating a
linear binding response with decreasing percentages of infected
cells, as measured with a gamma counter. Next we measured
gamma counts from equal aliquots of cryopreserved and thawed
splenocytes and lymph node cells, collected at necropsy from
uninfected and SIV-infected animals (Supplementary Fig. 1b).
The data demonstrate that even after a freeze-thaw cycle, the
7D3-PEG-64Cu-DOTA probe bound specifically to infected
cells. Subsequently, we performed a competition assay whereby
‘cold’ 7D3-PEG-DOTA was used to compete with bound ‘hot’
7D3-PEG-64Cu-DOTA from SIV1C and Hut78 cells, demonstrating epitope-specific binding of the probe (Supplementary Fig. 1c).
In order to assess differences between 7D3-PEG-DOTA– and
7D3-PEG-DyLight 650–labeled probes (used in flow cyto
metry assessments), we used both probes to label SIV1C cells,
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Control 1 with modified 7D3
Figure 1 | PET/CT results from uninfected
a
b
control and four chronically SIV infected,
viremic rhesus macaques. (a,b) Frontal,
sagittal and axial views and magnifications
Axillary LN
of a viremic monkey, Viremic 1, and of
Axillary LN
a representative uninfected monkey,
Control 1, imaged with the modified
5.6
7.8
Inguinal LN
7D3 probe. Sites of axial sections are
3.27
4.5
Inguinal LN
marked by a yellow line within the
1.95
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sagittal view. Scale bars (white): axial
and frontal view of torso and sagittal
0.85
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view of head, 100 mm; sagittal view of
whole body, 50 mm; lymph nodes (LN)
0
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Genital tract
and genital tract, 20 mm. (c,d) IHC
against SIV Gag in tissues from
PCR
c
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PET SUVmax, viremic
Viremic 1 (c) and in spleen from an
PET SUVmax, combined
10,000
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SIV-infected monkey with high viremia
controls
4.00
and in uninfected colon control tissues (d).
3.50
Arrows indicate infected mononuclear
1,000
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cells. Scale bars: colon uninfected (d),
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Jejunum
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50 µm; all other images (c,d), 20 µm.
100
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(e) Quantification of the PET data from
1.00
various tissues from 4 SIV-infected and
0.50
viremic monkeys (magenta) as well
0
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as 2 uninfected monkeys administered the
Colon uninfected
7D3 probe and 2 monkeys administered
Inguinal LN
Axillary LN
an isotype IgG probe (blue). The maximum
Chronic viremic
Ratio chronic viremic SUV
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standard uptake value (SUVmax) within
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the qRT-PCR results. Mean ± s.d. shown for
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in counts per min (CPM) adjusted for tissue
weight and the activity of the injected probe.
(g) Comparison of SUVmax results in the same
viremic and uninfected monkeys as in e;
a repeated measures ANOVA confirmed that the animal conditions were statistically different (P = 7.39 × 10−6). SUVmax values obtained with nonspecific
IgG control in uninfected animals are also included. (h) Measurement of the SUV ratio at two time points following probe injection (PI, post-injection).
demonstrating specific binding (Supplementary Fig. 1e). This
experiment showed that the fluorescent probe binds with the
same efficacy as the 7D3-PEG-DOTA (cold) probe. We then
assessed the DyLight 650–labeled probe, using flow cytometry
for its binding to SIV1C cells in the presence of serum and
SIV-specific antibodies generated during infection
(Supplementary Fig. 1f). Although there was less binding of
7D3 in the presence of the pre- and post-infection serum than
with monkey serum absent, we noted specific binding to SIV1C
relative to uninfected Hut78 cells.
Characterization of chronic SIV infection
To generate images of SIV dissemination in vivo, we injected the
7D3-PEG-64Cu-DOTA probe (labeled with 1–3.5 mCi/mg 64Cu)
into rhesus macaques intravenously. We tested probe stability by
pelleting virus from the plasma of the infected and uninfected animals and measuring the radioactivity (Supplementary Fig. 1d).
These data demonstrate that specific probe binding to virus was
still possible 24 h after injection of the probe. We found PET
and computed tomography (CT) imaging (hereafter PET/CT)
in both viremic and control animals to be optimal 24–36 h
after injection (data not shown). We then imaged macaques
chronically infected with SIVMAC239 and uninfected control
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ART 1 before ART
ART 1 during ART
ART 1 before ART
ART 1 during ART
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Figure 2 | PET/CT results from a chronically infected macaque, before and at 5 weeks of ART. (a) Standard uptake value (SUV) maps of GI tract,
lymph nodes (LN), genital tract, spleen and small bowel. NALT, nasal-associated lymphoid tissue. Arrows indicate areas for which specific PET signals
decreased during ART. Scale bars: frontal view of torso, 100 mm; sagittal view of head, 50 mm; LN and genital tract, 20 mm; small bowel, 15 mm.
(b) SUVmax values before and after 5 weeks of ART compared with background uptake in 2 uninfected animals. (c) qRT-PCR verification of residual virus
compared with SUVmax PET data at 5 weeks of ART in one representative treated macaque.
animals after injection with either a 64Cu-labeled, PEG-modified
7D3 mAb or a similarly labeled control IgG to test the ability of the
modified mAb to specifically target and detect SIV-infected cells
and tissues in vivo, displaying frontal, sagittal and axial images
(Fig. 1a,b, Supplementary Fig. 2a–f and Supplementary Table 1).
We generated standardized uptake value (SUV) maps for
each PET and corresponding CT image using OsiriX software
(Fig. 1a,b and Supplementary Fig. 2a–f). SUV is essentially the
measured activity within a region of interest, normalized by the
radioactive dose divided by the subject’s weight. In the viremic
animals, SUV values greater than those observed in the control
cases were localized to the gastrointestinal (GI) system, specifically within the ileum, jejunum and colon, and the axillary
and inguinal lymph nodes, correlating with previously reported
findings12,19–21. We also detected uptake within the lungs, a less
well-characterized site of viral infection22. As can be seen in detail
in slice sequences for monkeys Viremic 1 and 2, the uptake followed the contours of the ileum and colon (Supplementary
Fig. 3a,b). We also consistently detected antibody uptake within
the nasal cavity, likely reflective of the nasal-associated lymphoid tissue (NALT) or nasal turbinates (Fig. 1a), an area that
has received little attention as a source of viral replication. In
males, we frequently observed uptake within the genital tract,
specifically in the vas deferens and epididymis, corroborating previous reports23–25. In contrast to a previous report26, infection of
epithelial cells was not detected in the male genital organs. In the
uninfected monkeys, background was evident within the liver, heart,
kidneys and spleen, which is typical of antibody-based radio
tracers17,18. It should be noted, however, that SUVs were significantly higher in infected animals than in control cases in each of these
organ systems, indicating specific uptake (P < 0.05, Kruskal-Wallis
test; Supplementary Table 2). A sequence of frontal slice images
obtained on the uninfected monkey Control 1 after administration
of 64Cu-7D3 demonstrates that uptake was minimal throughout
the GI tract and that background was restricted to the heart, liver,
kidneys and spleen (Supplementary Fig. 3c).
PET quantification and comparison with qRT-PCR and IHC
We verified the imaging signal data by qRT-PCR and through
the examination of sections of the specific tissue of interest using
immunohistochemistry (IHC) for SIV Gag protein (Fig. 1c,d and
Supplementary Fig. 2). These confirmatory studies used rectal
biopsy tissues obtained immediately after imaging and/or tissues
obtained post-mortem. On the basis of the IHC data, the GI tract,
lymph nodes and spleen all contained infiltrating SIV-infected
cells of lymphocyte or macrophage morphology. Negative control
tissue (Fig. 1d) did not contain any detectable signal using the
same IHC protocol. We also performed qRT-PCR using tissue
samples from the colon, small bowel, spleen and inguinal and
axillary lymph nodes for all of the chronically infected animals
and controls (Fig. 1e). Corroborating our IHC results, we detected
viral RNA in all cases, with the highest levels in the colon; we did
not detect RNA in the controls. In addition, we quantified the
64Cu-radioactivity associated with aliquots of rectal biopsies from
viremic monkeys Viremic 1 and 2 and uninfected Controls 1 and 2
with a gamma counter. The signal from these tissues, normalized
for the mass of the biopsy and the total amount of radioactivity
administered, was 17.6 times higher on average in infected
animals (Fig. 1f) than in both controls, providing additional
confirmation of the specificity of the PET imaging.
To compare the PET results from viremic animals to those
of the controls, we quantified the data using SUV (Fig. 1a,b,e,g
and Supplementary Fig. 2). We chose volumes of interest in the
PET/CT fusion images by outlining the organ manually in the CT
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Figure 3 | PET/CT results from SIV-infected elite controllers (ECs). (a) Frontal, sagittal and axial views and magnifications from one SIV-infected
EC macaque 36 h after injection of the labeled antibody. The axial section is denoted by a yellow line within the sagittal view. Macaque EC 1 was
infected for over 6 years and displayed plasma viral loads of fewer than 60 copies of viral RNA per ml for the last 5 years. Scale bars (white): axial and
frontal view of torso and sagittal view of head, 100 mm; sagittal view of whole body, 50 mm; lymph nodes (LN) and genital tract, 20 mm. (b) SUVmax
quantification results from the PET/CT imaging, comparing 4 viremic and 4 EC monkeys with 2 uninfected controls (mean ± s.d.). See Supplementary
Note for a description of the statistical analysis of this data. (c) IHC results against the SIVMAC239 Gag for macaque EC 1. Arrows indicate infected
mononuclear cells in select tissue biopsies. Scale bar, 20 µm.
images throughout the image slices. Using the organ volume as
the region of interest (ROI), we then determined the maximum
SUV within that organ and compared the SUVmax within viremic
and uninfected animals with the qRT-PCR results from the corresponding colon, small bowel, spleen and inguinal and axillary
lymph nodes of the same animal (Fig. 1e). These data suggest that
the PET SUVmax values mimic the general trends of the PCR data.
For the spleen, which tends to have higher background uptake,
the SUVmax minus background is a more relevant comparison
with the qRT-PCR data. It should be pointed out that the SUV
data are unlikely to match the PCR data precisely, as protein and
RNA expression levels may differ.
Next, to address the specificity of the PET signals, we
compared the SUVmax measurements for various tissues from
chronically infected and uninfected macaques injected with
either the modified 7D3-labeled antibody or with a labeled
isotype control antibody (Fig. 1g and Supplementary Fig. 2g).
The data suggest that there is increased uptake within organ
systems likely to contain virus or virally infected cells and
tissue. For each group of animals, we then performed an overall
comparison of the PET measurements that included the signals
of all organs, applying a fully nested hierarchical ANOVA model
for the SUVmax response. The null hypothesis for the comparison
was that the viral status of the animal and the injected probe
would not influence the imaging results. We found that the
viremic animals were significantly different from the uninfected
controls (P = 7.39 × 10−6). However, animals within each group,
infected or uninfected, were not significantly different from each
other (P = 0.89). The analysis also showed that both the infection status (animal group) and organs contributed significantly
to the SUVmax value. We found that for the chronically viremic
animals and both control groups, the measurements for each
organ within each group were significantly different from each
other, yielding P values of 1.4 × 10−9, 7.1 × 10−27 and 4.78 × 10−19,
respectively. Furthermore, when we applied the Kruskal-Wallis
test for each organ separately (Supplementary Table 2), the signals
measured in viremic and uninfected controls were statistically
distinct except for in muscle.
Another method of assessing specific uptake for a particular
organ is to examine the dynamics of uptake. Owing to logistics,
we scanned animals at 12, 24 and/or 36 h after injection and plotted the ratios of the average SUVmax values at each time point for
each organ (Fig. 1h). When we compared the viremic monkeys
with the aviremic controls, all of the ratios in viremic animals
were higher—typically above 0.6, with the GI tract giving
values >1.0—indicating continued specific uptake of the probe. The
decay rate and SUVmax values in whole blood (SUVmax ≈ 0.3–0.4;
data not shown) were similar to those of muscle, and remained
similar between groups. We did not detect uptake in the
central nervous system, probably because of probe exclusion by
the blood-brain barrier27.
SIV localization before and during antiretroviral therapy
In order to confirm the sensitivity of this method and its ability to
track SIV replication anatomically during treatment, we first imaged
three chronically infected animals (ART 1, ART 2 and ART 3)
36 h after injection with our modified 7D3 probe and then initiated
them on ART (20 mg PMPA (9-(2-phosphonomethoxypropyl)
adenine) per kg body weight per day (mg per kg per d) and
50 mg per kg per d emtricitabine (FTC) each subcutaneously
and 100 mg/d for 40 d of integrase inhibitor L-870812) (Fig. 2
and Supplementary Fig. 4). All three animals were aviremic by
SIV localization in elite controllers
We then applied the methodology to SIV-infected ECs. ECs are
individuals that naturally suppress SIV (or HIV) replication to
undetectable levels in plasma for extended periods of time without antiretroviral intervention1,2. Given the challenges studying
viral persistence in these animals, they were an ideal test for our
approach. EC monkeys exhibited detectable uptake (Fig. 3 and
Supplementary Fig. 7) within the GI tract, genital tract, NALT,
lungs, spleen and axillary lymph nodes. These imaging data were
supported by IHC in biopsy samples (Supplementary Fig. 7,
SUV
EC
C
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re oni
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z = –352.5 mm
d
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EC
3–4 weeks of treatment (having viral loads <60 copies of RNA
per ml; Supplementary Fig. 5) and imaged again after 5 weeks
on ART (Fig. 2 and Supplementary Fig. 4). Prior to treatment,
there was measurable SIV signal localized within the GI tract,
NALT, genital tract and axillary and inguinal lymphoid tissue.
After 34 d of treatment, all organ systems exhibited decreased
uptake (Fig. 2a,b, Supplementary Fig. 4 and Supplementary
Table 3a). However, there was residual signal (above the background) in all organ systems, with moderate SUVmax values
still remaining in the colon, spleen, male genital tract, NALT and
individual lymph nodes for specific animals. In none of the cases
did the SUVmax decrease to our measurable limit (background).
To assess the statistical significance of the SUVmax measurements, we performed a hierarchical ANOVA analysis as described
above. The differences between the SUVmax data before and after
treatment from all of the organs imaged were significant, with a
P value of 0. In a pairwise comparison, ART 1 and ART 3 were
significantly different from ART 2 (P = 0.0027), demonstrating
the individual variation in ART treatment.
To verify the imaging results, we performed qRT-PCR on
multiple tissue samples and compared the data directly with
SUVmax data (Fig. 2c and Supplementary Fig. 4c). The samples
included colon, small bowel, spleen, and right and left inguinal and
axillary lymph nodes; these were collected at necropsy performed
after 39 or 40 d on ART. Even though our PET procedure measures Env protein and qRT-PCR detects viral RNA, residual virus
or infected cells were indeed present in the locations identified by
PET. Both the spatial variation within an animal and the variation
between animals suggested by PET was confirmed with qRT-PCR
data (Supplementary Fig. 6 and Supplementary Table 3b), with
two-orders-of-magnitude variation within an organ and between
animals. Additionally, the nasal turbinate, genital tract and lung
samples were all positive for viral RNA (Supplementary Table 3b),
indicating virus localization during both chronic and treated
conditions, a similar result to that observed in viremic animals.
a
Angular second
moment
Figure 4 | Comparison of viremic with elite controller (EC) macaques.
(a) Comparison of SUVmean and SUV voxel fraction (fraction of total
volume of GI tract) within the GI tract of 4 chronic SIV-positive and 4 EC
macaques. The voxel fractions that contained SUVs above 1 and below 3.3
(excludes interfering signals) were included. (b) Haralick texture function
measurements of angular second moment and contrast for regions of
interest (ROIs; depicted by red boxes in c,d). The angular second moment
is a measure of homogeneity, whereas the contrast metric represents the
local variations within an image or ROI. Data reported as mean ± s.d.
(c,d) Representative axial cross sections of a chronically infected macaque
(Viremic 1) and one controller macaque (EC 2). Red arrows indicate uptake
within the liver; white arrows in c point toward the GI tract; white arrows
in d point to mesenteric lymph nodes and other foci. Scale bars, 50 mm.
Voxel fraction with GI signal
above threshold
Articles
EC 2
z = –390.5 mm
4.27
3.3
1.75
0.95
z = –432.5 mm
0
Supplementary Table 2 and Supplementary Note). In ECs,
the uptake was restricted to smaller regions or foci as compared to viremic animals. When quantified, the SUVmax organ
signal appeared to approximate the results in viremic animals
(Fig. 3b). However, when we applied a hierarchical ANOVA,
we found that overall the PET SUVmax data for the viremic
animals were statistically distinct from those of the ECs and
the control animals (P = 2.78 × 10−5). To clarify the differences
between the ECs and viremic animals, we measured the SUVmean
within the GI tract and compared the voxel fractions (fraction
of total volume of GI tract) (Fig. 4 and Supplementary Fig. 8).
The GI tract values in the viremic animals were 2.1 and 6.38 times
greater than in the EC animals for SUVmean and voxel fraction,
respectively. Thus, although the viremic macaques and ECs
had regions of comparably high uptake, in ECs, this was
spatially restricted to much smaller volumes, and therefore the
overall probe uptake was lower. We calculated additional
metrics quantifying the spatial distributions within the GI
tract, which further supported this conclusion (Fig. 4a,b and
Supplementary Note).
DISCUSSION
We describe the development of a non-invasive, sensitive immuno
PET radiotracer and an approach to define the localization of
SIV-infected tissue and free virus within live, chronically viremic,
ART-treated and EC animals. The method can be repeated within
the same animals (for example, before and during ART) without
any adverse effect. In viremic animals, infection was concentrated
within the mucosa of the gut, reiterating that these tissues are a
major site of SIV replication12,20,21. However, we also observed
discrete areas of virus replication, confirmed by qRT-PCR and
IHC, both in nasal-associated tissues (post-ART) and in the
reproductive tract of male animals. Within chronically infected,
aviremic, ART-treated as well as EC animals, the methodology
was able to detect residual virus, corroborated by qRT-PCR data.
Thus, this approach provides the ability to identify novel areas of
virus replication that may otherwise be difficult to sample in live
animals. It may also provide a powerful tool to monitor the kinetics
of viral replication in tissues over time during the application of
novel therapeutic approaches. With the current efforts toward
HIV eradication or functional cure, we believe that this method
can be useful for determining organ-specific efficacy, which is
crucial to the elimination of virally infected cells.
Articles
Our data also indicate that care must be taken when analyzing
biopsies from aviremic subjects, especially ART-treated subjects,
which may result in erroneous conclusions due to sampling
(Supplementary Fig. 6 and Supplementary Table 3b). The
detailed study of the cellular composition of these specific foci
of infection, combined with site-specific drug metabolite levels
and aided by the ability to image these specific sites, will likely be
key to the development of directed therapies aimed at clearing
infection from these sites in both controllers and individuals
under ART28–30.
Although additional refinements to improve contrast and
uptake are ongoing, we think that the methodology should be
translatable to humans in the future because of the availability of
anti-Env HIV antibodies31 and because the imaging approach is
based on technologies already used in the clinic17,18. It is applicable for studies investigating the eradication of HIV infection and
targeting of virus reservoirs28,32. Moreover, use of this technology
during acute SIV infection may provide improved delineation of
spatial kinetics of viral spread based on the route of infection and
allow the identification of stages at which interruption of infection
may be targeted using prophylactic methods33.
Methods
Methods and any associated references are available in the online
version of the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank the animal and care staff of the Yerkes National Primate Research
Center; B. Lawson, E. Strobert, J. Wood, S. Jean and M. Thompson; and
D. Votaw and the Emory Clinic imaging team for their help in the conduct of
these studies. Funding for these studies was provided by a developmental grant
from the Yerkes National Primate Research Center, the Georgia Research Alliance,
US National Institute of Allergy and Infectious Diseases grants R21-AI095129
to P.J.S. and F.V., R01 AI078775 and 8R24 OD010947 to F.V. and R01 AI078773
to A.A.A., the Center for AIDS Research P30 AI050409 and the base grant P51
OD11132 in support of Yerkes. E.H. is supported by a Georgia Research Alliance
Eminent scholarship. We also thank M. Miller (Gilead Inc.) for the PMPA and FTC
and D. Hazuda (Merck) for the integrase inhibitor. We gratefully acknowledge the
help, support, encouragement and advice of O. Sharma (NIAID) given during the
course of these studies.
AUTHOR CONTRIBUTIONS
P.J.S., E.H. and F.V. conceived of the project. C.Z. produced the imaging
agent, and P.J.S. and F.V. performed the radioactive labeling. P.J.S., F.V., C.Z.,
E.L.B., K.A.R., S.G., K.S., F.C.-S., P.K.A., J.J.H. and S.N.B. performed experiments.
A.A.A. contributed animals and reagents. E.H. and J.A.H. provided reagents and
advice. D.M.S. assisted with PET imaging. B.V. performed the statistical analyses.
P.J.S., F.V., A.A.A. and E.H. wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the
online version of the paper.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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ONLINE METHODS
Antibody production. The monoclonal antibody secreting hybridoma (clone 7D3) was provided by J.H.13. Preparation and purification of 100 mg of 7D3 was performed by R. Mittler of the Emory
CFAR Immunology Core using standard methods. The isotype
control antibody was purchased from Innovative Research,
catalog # IR-MS-GF-ED (Endotoxin Depleted Mouse IgG).
Antibody modification for in vivo imaging. To conjugate 1,4,7,
10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (DOTA
NHS) and poly(ethylene glycol) esters (PEG ester) to surface
lysine residues of 7D3, molar ratios of 60:1 and 20:1, respectively,
were used. Briefly, 1 mg of clone 7D3 mAb (at 3.8 mg/ml) was
buffer exchanged with 0.1 M phosphate buffer (EMS), pH 7.3,
plus Chelex 100 (Bio-Rad) using a 10-kDa Amicon spin column
(Millipore). Then 1 µl of 0.5 M DOTA NHS ester (Macrocyclics)
in phosphate buffer and Chelex 100 and 32 µl of 5 mM m-PEGSMB 10K (Nectar), also in phosphate buffer and Chelex 100,
were added and reacted for 4 h at RT on a rotator. Unconjugated
reagents were removed using 30-kDa Amicon spin columns in
phosphate buffer plus Chelex 100. Samples were quantified via
UV-VIS spectroscopy, and the conjugations verified via gel electrophoresis using Tris-acetate gels in TA running buffer with SDS
(Invitrogen). The modified 7D3 antibody was then divided into
aliquots and lyophilized for storage.
Radiolabeling. Lyophilized PEG-DOTA-7D3 was resuspended
in Chelexed 0.1 M NH4OAc, pH 5.5 (Sigma). Copper (II)-64
chloride (Washington University) was diluted similarly; they
were then mixed together at a ratio of approximately 5 mCi/mg
and incubated at 37 °C for 1 h. The antibody conjugates typically
labeled in the range of 1–3.5 mCi/mg per dose. Each dose was
buffer exchanged with pharmaceutical-grade saline three times
using a 10-kDa centrifugal filter to a final volume of 20 µl. The
conjugated mAb was then added to 1 ml of pharmaceutical-grade
sterile saline in a sterile glass vial. Uptake was confirmed on an
aliquot using thin layer chromatography. The labeled antibody
conjugates gave values in the range of 1–3.5 mCi/mg.
In vitro 7D3-PEG-64Cu-Dota probe specificity test. SIV1C cells
with one integrated copy of SIVmac251 and uninfected parental
Hut78 cells (ATCC TIB-161) were cultured in RPMI 1640
supplemented with penicillin-streptomycin and 10% FBS. 24 h
before assaying, the cell cultures were supplied with 100 U/ml of
rMamu IL-2-Fc to induce increased expression of SIV Gp120 on
the surface of SIV1C cells34. For staining, tubes were set up with
500,000 cells containing varying mixtures of SIV1C and Hut78
cells (100%, 75%, 50%, 25% and 0% SIV1C). 200 ng of freshly
radiolabeled 7D3-PEG-64Cu-DOTA were added to each tube of
cells suspended in 200 µl of PBS and incubated for 15 min at room
temperature. The tubes were then washed three times with 2 ml
of PBS and read in a Packard gamma counter. All cell lines used
were free of mycoplasma.
Competition of ‘cold’ 7D3-PEG-DOTA with 7D3-PEG-64
Cu-DOTA. 250,000 SIV-1C or control Hut78 cells were suspended
in tubes with 200 ng of radiolabeled 7D3-PEG-64Cu-DOTA
followed by the immediate addition of varying amounts of cold
7D3-PEG-DOTA (0, 56.3, 237.5, 950 and 3,800 ng). Incubation,
doi:10.1038/nmeth.3320
washing and reading of the tubes radioactivity were done as
described above.
Ex vivo labeling of primary cells with 7D3-PEG- 64Cu-DOTA.
Cryopreserved lymph node cells and splenocytes from both
SIV-positive and SIV-negative rhesus macaques collected at
necropsy were thawed, washed in PBS and counted. 5 million
cells for each tissue were suspended in 200 µl PBS and 500 ng
of 7D3-PEG-64Cu-DOTA, and 10 µl of serum from an SIV
negative animal that were heat inactivated for 30 min at 56 °C.
The cells were then incubated for 15 min at room temperature
followed by four washes with 2 ml of PBS and read in a Packard
gamma counter.
Detection of 7D3-PEG-64Cu-DOTA on viral pellets. At the time
of imaging, blood was collected in citrate CPT tubes, which were
processed according to vendor instructions (BD Biosciences).
1 ml of the resulting plasma was centrifuged at 21,130g for 1 h at
4 °C to pellet virus. The plasma was removed, and the pellet was
washed with 1 ml of PBS twice and centrifuged again. After the
final wash the pellet read in a gamma counter.
In vitro assay for serum modulation of probe binding. Aliquots
of 500,000 SIV1C cells or Hut78 cells were incubated at room
temperature for 15 min with 200 ng of 7D3-PEG-DyLight 650 and
50 µl of PBS, SIV pre-infection or SIV post-infection serum. Cells
were then washed twice with 2 ml of PBS with 2% FBS. Cells were
fixed with 200 µl of 1% PFA in PBS and read on an LSR-II flow
cytometer (BD). The data were analyzed using FlowJo software
(Tree Star).
Immunofluorescence detection of probe binding. The ability of
the 7D3-PEG-DOTA to bind Gp120 was tested via immunofluorescence in SIV1C (SIV positive)35 or control Hut78 cells. Cells
were cytospun onto glass slides, fixed and permeabilized in 50%
methanol/acetone for 10 min at −20 °C, blocked with 5% BSA and
stained using 10 ng/µl PEG-DOTA-7D3 and a Cy3-conjugated
AffiniPure Donkey anti-mouse secondary antibody (#715-165-150,
Jackson ImmunoResearch). The 7D3-PEG-650 used for flow
cytometry measurements was also tested via immunofluorescence using the same protocol and showed a similar ability to
bind Gp120.
Probe modification for fluorescence measurements. In order
to conjugate PEG ester and DyLight 650 NHS ester (Thermo
Scientific) to surface lysine residues of 7D3, 20:1 and 4:1
molar ratios, respectively, were used. Briefly, 1 mg of 7D3 was
buffer exchanged with 0.1 M phosphate buffer using a 10-Da
Amicon spin column. 32 µl of 5 mM PEG and 1.12 µl of 25 mM
DyLight 650 were added and reacted for 2 h at RT on a rotator.
Unconjugated reagents were removed using 30-Da Amicon spin
columns in phosphate buffer. Samples were quantified via UV-Vis
spectroscopy and stored at 4 °C.
Monkeys. A total of 14 adult Indian rhesus macaques age 5–11
years were used in this study as outlined in Supplementary Table 1.
All animals were born at Yerkes National Primate Research Center
and were maintained at the Yerkes National Primate Research
Center of Emory University in accordance with the regulations of
nature methods
the Guide for the Care and Use of Laboratory Animal, 8th edition.
The experiments were approved by the Emory Institutional Animal
Care and Use Committee as well as biosafety review board. Each
of the animals used in the studies reported herein was inoculated
with 200 TCID50 (50% tissue culture infective dose) of SIVMAC239
intravenously and served as a source for blood and tissues at various time points post-infection. All animals were typed for MAMUA*001, MAMU-B*008 and MAMU-B*017 alleles (Supplementary
Table 1), and their plasma viral loads determined by qRT-PCR
by the Emory CFAR Virology Core36,37. Of note, none of the EC
monkeys expressed MAMU-B*008 or MAMU-B*017, traditionally
associated with increased control of viremia38,39.
PET/CT imaging. Animals were imaged using a Siemens
Biograph 40 PET/CT, using image settings for 64Cu. Typically
between 250 and 300 slices were compiled for each macaque
depending on body size.
Necropsy. Animals showing signs of disease or distress or that
reached IACUC end points, such as pain or stress that could
not be alleviated using standard analgesics and/or chemotherapy were humanely euthanized with an intravenous overdose
of pentobarbital sodium according to the guidelines of the
American Veterinary Medical Association. A complete necropsy
was performed on these animals. For histopathologic or immunohistochemical examination, various tissue samples were fixed
overnight in 4% paraformaldehyde, embedded in paraffin and
sectioned at 4 µm.
Biopsy. Animals were anesthetized with either ketamine (5–10 mg
per kg body weight (mg per kg), intramuscular) or Telazol
(3–5 mg per kg, intramuscular) and placed in ventral or lateral
recumbency, and a lubricated anoscope was introduced into the
rectum. Feces were removed using clean cotton tipped applicators.
Up to 20 colorectal biopsies per collection were obtained using
a biopsy forceps under visualization. The procedure is generally
well tolerated and does not require analgesics. The colorectal
biopsies were collected in RPMI 1640 with gentamicin at 4 °C.
They were then removed from media weight in tared tubes and
read on the Packard gamma counter.
PET image analysis. PET/CT fusions were analyzed using OsiriX
software on a MacBook Pro or Mac Pro. The volume ROI tool
was extensively used to measure standard uptake values (SUVs),
SUVmax and SUVmean within a specific organ. In order to evaluate
the voxel fraction with uptake, the 3D images from OsiriX were
exported as TIFF stacks and imported into PerkinElmer Volocity
software. Within Volocity, 3D ROIs were created to represent the
GI tract; the mean uptake of the GI tract was measured, the total
volume, as well as the voxel fraction (volume fraction) of objects
with SUV values between 1.0 and 3.3 within the GI tract. For each
imaging experiment presented, SUVs were compared at the same
time point injection of the radiotracer. Haralick texture features40
were computed using an ImageJ plug-in or in Matlab.
Statistical methods. This manuscript describes three experiments:
(i) a comparison of PET SUVmax measurements in chronically
infected macaques and uninfected macaques and measurements
in uninfected macaques using an isotype control modified
nature methods
antibody; (ii) a comparison of SUVmax measurements in three
animals, all chronically infected with SIV, imaged with PET, treated
with ART for 4 weeks and then imaged again via PET; and (iii)
PET imaging in ECs (>5 years aviremic), chronically infected animals and uninfected control animals. The results from these three
experiments have been analyzed using two statistical approaches.
First, a global comparison of the PET measurements for each
animal state was performed that included the measurements of
all of the organs. This was achieved applying a fully nested hierarchical ANOVA model (implemented in Matlab (MathWorks))
for the SUVmax response yijk = µ + αi + βj(i) + γk(j) + εijk, where yijk
is the SUVmax, α is the effect of the infection status, β the effect
of animal nested in status, and γ is the effect of the organ system
nested in each animal. In the model discussed above, Q-Q plots
of residuals of the proposed model against Gaussian quantiles for
each experiment exhibited a linear pattern clearly indicating that
the assumption of normality of the SUVmax measurements was
appropriate. In addition, a Kruskal-Wallis, Bonferroni-ANOVA,
or Wilcoxon–Mann-Whitney test was applied, which enabled the
analysis of each organ separately comparing the differences in that
organ’s measurement for each animal state.
Sample size and exclusions. Groups of 4 monkeys were used for
analyses, with viral loads in comparable ranges. For the ART group,
3 animals are shown (a fourth developed AIDS during treatment
and was euthanized). However, these comparisons are within the
same animal before and after therapy. A total of 8 viremic monkeys
were tested (including pre-ART values), 4 elite controllers, and
2–4 uninfected controls for background values. (Supplementary
Table 1). No sample-size estimates were performed to ensure
adequate power to detect a prespecified effect size.
Randomization and blinding. The grouping was based on plasma
viral loads (viremic: 3 × 104 to ~3 × 106 viral rRNA copies per
ml of plasma; ECs and ART treated: <60 copies vRNA per ml of
plasma). No other randomization was utilized. Male and female
monkeys were utilized (Supplementary Table 1). No blinding
was utilized.
Quantitative RT-PCR. Plasma and tissue RNA was isolated using
the QiaAmp Viral RNA Mini-Kit and RNeasy Mini Kit, respectively
(Qiagen). Viral RNA (vRNA) levels were measured by qRT-PCR;
the assay sensitivity was 60 vRNA copies per ml for plasma, and
RNA copy numbers in tissues were normalized per ng of total
RNA and represented as SIV RNA copies per ng of total RNA.
IHC in tissue sections. Immunohistochemical staining was
performed using an alkaline phosphatase–streptavidin–biotin
procedure. Tissues collected by biopsy/necropsy were fixed
overnight in 4% paraformaldehyde, embedded in paraffin and
sectioned at 4 µm. Paraffin-embedded sections were subjected
to deparaffinization in xylene, rehydration in graded series of
ethanol, and rinsed with distilled water. Antigen retrieval was
performed by immersing sections in a target retrieval solution
(Dako) at 120 °C for 30 s in a steam pressure decloaking chamber
(Biocare Medical). The slides were then allowed to cool for
20 min, washed with distilled water, and placed in Tris buffer
saline (Sigma) for 5 min. Sections were then blocked with Protein
Block, Serum-Free (Dako) for 15 min. The primary monoclonal
antibody (SIVmac p27 monoclonal antibody (FA2), NIH AIDS
Reagents Program) was diluted in antibody diluent (Dako) at
doi:10.1038/nmeth.3320
34. Lairmore, M.D., Post, A.A., Goldsmith, C.S. & Folks, T.M. Cytokine
enhancement of simian immunodeficiency virus (SIV/mac) from a
chronically infected cloned T-cell line (HuT-78). Arch. Virol. 121, 43–53
(1991).
35. Daniel, M.D. et al. Isolation of T-cell tropic HTLV-III-like retrovirus from
macaques. Science 228, 1201–1204 (1985).
36. Hong, J.J., Amancha, P.K., Rogers, K., Ansari, A.A. & Villinger, F. Spatial
alterations between CD4+ T follicular helper, B, and CD8+ T cells during
simian immunodeficiency virus infection: T/B cell homeostasis, activation,
and potential mechanism for viral escape. J. Immunol. 188, 3247–3256
(2012).
37. Hong, J.J., Amancha, P.K., Rogers, K., Ansari, A.A. & Villinger, F.
Re-evaluation of PD-1 expression by T cells as a marker for immune
exhaustion during SIV infection. PLoS ONE 8, e60186 (2013).
38. Torriani, M. et al. Increased FDG uptake in association with reduced
extremity fat in HIV patients. Antivir. Ther. 18, 243–248 (2013).
39. Yant, L.J. et al. The high-frequency major histocompatibility complex class
I allele Mamu-B*17 is associated with control of simian immunodeficiency
virus SIVmac239 replication. J. Virol. 80, 5074–5077 (2006).
40. Haralick, R.M., Shanmugam, K. & Dinstein, I. Textural features for image
classification. IEEE Trans. Syst. Man Cybern. 3, 610–621 (1973).
room temperature. The sections were incubated with the primary
mAb overnight in a humidified chamber at 4 °C followed by
biotinylated rabbit anti-mouse antibody (#E035401-2, Dako) and
alkaline phosphatase–streptavidin conjugate (#S-921, Invitrogen)
for 30 min each at room temperature. Following each incubation, the slides were washed with Tris buffer saline containing
Tween 20 (Sigma). Antibody labeling was visualized by
development of the chromogen (Vulcan Fast Red; Biocare
Medical). The reaction was stopped by immersing the slides
in distilled water, and nuclei were counterstained using Gill’s
hematoxylin. Digital images of Vulcan Fast Red–stained slides
were captured at 400× magnification with an Olympus BX43
microscope equipped with a digital camera (DP26, Olympus)
using cellSens digital imaging software 1.9 (Olympus). Positive
controls included samples of spleen and lymph node from
SIV-infected rhesus macaques. Negative control, colon, was
performed by omitting the primary mAb.
doi:10.1038/nmeth.3320
nature methods
errata
Erratum: Whole-body immunoPET reveals active SIV dynamics in
viremic and antiretroviral therapy–treated macaques
Philip J Santangelo, Kenneth A Rogers, Chiara Zurla, Emmeline L Blanchard, Sanjeev Gumber, Karen Strait, Fawn Connor-Stroud,
David M Schuster, Praveen K Amancha, Jung Joo Hong, Siddappa N Byrareddy, James A Hoxie, Brani Vidakovic, Aftab A Ansari,
Eric Hunter & Francois Villinger
Nat. Methods 12, 427–432 (2015); published online 9 March 2015; corrected after print 11 June 2015
npg
In the version of this article initially published, the first name of Emmeline L. Blanchard was misspelled as Emeline. The error has been
corrected in the HTML and PDF versions of the article.
nature methods
Supplementary Figure 1
In vitro binding of modified mAb clone 7D3 to SIV gp120, interrogated by scintillation counter, fluorescence microscopy and flow
cytometry.
a. Counts per minute from 7D3-PEG-64Cu-DOTA labeled SIV-1C serially diluted with Hut78 (non-infected) cells,
demonstrating a linear response in samples with equal cell numbers. b. Counts per minute from cryopreserved and
thawed splenocytes and lymph node cells, originating from uninfected and SIV infected animals collected at necropsy.
The data demonstrates that even after a freeze-thaw cycle the 7D3-PEG- 64Cu-DOTA probe binds specifically to infected
cells. Shown are means and standard deviations from lymph node and spleen cells from 2 and 1 uninfected controls and
3 and 5 infected monkeys, respectively. c. Competition assay where unlabeled “cold”-7D3 was used to compete 200 ng
bound “hot” 7D3-PEG-64Cu-DOTA from SIV-1C and Hut78 cells, demonstrating epitope-specific binding of the probe. d.
Probe bound virus pelleted from infected (Viremic 1 and Viremic 2) versus non-infected (Control 1 and Control 2) animal
plasmas, 24 hrs post contrast agent injection, measured in counts per minute normalized to the dose given to each
Nature Methods: doi:nmeth.3320
animal. This demonstrates that at 24 hrs post injection, the probe is still stable, and can bind to its target specifically. e.
Representative images of 7D3-PEG-DOTA (modified 7D3) and 7D3-PEG-Dylight 650 binding to SIV-1C cells,
demonstrating specific binding. No statistical difference was found between the two probes. No signal was detected for
either probe in Hut78 control cells (data not shown). This experiment demonstrates that the 650 labeled fluorescent probe
binds with the same efficacy than the 7D3-PEG-DOTA (cold) probe. f. Flow cytometry profiles of 7D3-PEG-Dylight 650
binding to control cells, SIV-1C cells, and SIV-1C cells in the presence of pre-infection and post-infection serum. While
both pre and post infection serum appear to compete with 7D3 binding to the infected cells, specific signal is still clearly
seen relative to binding to uninfected cells.
Additional PET and CT results from chronically SIV-infected viremic and control macaques.
a, b, c. Frontal, sagittal and axial views are presented, as well as magnified views (marked by a colored box and associated image
denoted by the same color outline) of regions of the frontal and sagittal sections. The site corresponding to the axial section is identified
within the sagittal view by a yellow line, while the sagittal view location, if not centerline, is denoted by a blue line in the frontal view.
Single plane PET/CT cross-sections from macaques, Viremic 2-4, (for plasma viral loads see Supplementary Table 1) scanned at 24
hrs post injection of the contrast agent. Both animals demonstrated PET signals within the GI tract, including the ileum, jejunum, and
colon, as well as within axillary and inguinal lymph nodes, and lungs. SIVgag p27 specific IHC for select organs of macaques Viremic 24, are also presented. Infected mononuclear cells (black arrows) were detected in the ileum, jejunum, and colon, as well as within
lymph nodes and the spleen, verifying the PET results. d. Single plane images of modified 7D3 within non-infected macaque, Control 2.
The sagittal image is taken from the centerline and the axial view is denoted by the yellow line within the sagittal view. The 7D3 probe
did not yield significant uptake within the gut, axillary, mesenchymal, or inguinal lymph nodes. Background uptake was highest within
the liver, heart, kidneys and spleen, as expected, though the signals measured in these organs were markedly lower than those
quantified from the same organs in SIV infected monkeys, suggesting specific uptake above background in the infected animals. e.
Control monkey number 1 given a modified isotype control IgG antibody; background signal detected was significantly lower than in the
infected animals, even within the liver, heart, and kidneys, sites for which background was expected. f. Single plane images of modified
mouse IgG within chronically-infected macaque, Viremic 5. Uptake was generally similar to the modified mouse IgG in the non-infected
animals. g. SUVmax quantification for the chronically infected and non-infected animals, with the isotype control probe (modified mouse
IgG) data in a chronically infected animal was added, demonstrating similar uptake to the non-infected animal.
Cross-sectional views of representative animals.
a. Cross-sections of the frontal torso and abdomen of SIV infected viremic monkey Viremic 2. Each slice is 1.37 mm thick; 28 slices are
displayed, amounting to a frontal view every 2.74 mm. A SIV positive signal is apparent throughout the gastrointestinal tract, especially
within the ileum, jejunum and colon, and within the lung. Background signal, as expected, was high within the liver, heart, and kidneys.
b. Cross-sections of the frontal torso and abdomen of SIV infected viremic monkey Viremic 1. Each slice is 1.37 mm thick; 24 slices are
displayed, amounting to a frontal view every 2.74 mm. A SIV positive signal is apparent throughout the GI tract and within the lung.
Background signal, as expected, was high within the liver, heart, and kidneys. c. Cross-sections of the frontal torso and abdomen of
uninfected monkey Control 1 given modified 7D3. In the frontal views, each slice is 0.98 mm thick, 24 slices are displayed, amounting to
a frontal view every 1.96 mm. Very little signal above muscle background within the gastrointestinal tract, and lymph nodes.
Background signal, as expected, was high within the liver, heart, and kidneys, and measurable within the lung.
PET-CT results from two additional chronically infected macaques, before and at 5 weeks of ART treatment.
PET/CT images of two SIV chronically infected macaques, ART 2 and 3, prior to and at 5 weeks of ART. a. Standard uptake value
(SUV) maps of GI tract, lymph nodes, genital tract, spleen and small bowel, demonstrating decreased probe uptake after 5weeks of
ART. b. SUVmax values before and after 5 weeks of ART, compared with background uptake in non-infected animals. c. qRT-PCR
verification of residual virus compared with SUVmax PET data..
10,000,000
Viral Load (RNA copies/ml)
ART 1
ART 2
ART 3
1,000,000
100,000
10,000
1,000
100
10
ART day 0
ART day 13
ART day 20
ART day 32
Kinetics of plasma viral loads in the SIV-infected monkeys ART 1, 2 and 3, before and during ART.
All 3 monkeys were subjected to PET/CT analysis prior to ART initiation and again after 34 days on ART, before being euthanized on
days 38/39 for tissue collections and analyses. By 3-4 weeks of ART, plasma viral loads were below the 60 copies/ml detection limit of
the qRT-PCR assay.
Tissue viral loads in SIV-infected, ART-treated monkeys.
a. The average tissue viral loads (SIV vRNA copies/ng total RNA) were generated from 6 separate spatial samples for the colon, 4 for
the jejunum and 4 for the ileum. The average viral loads were derived from the results of the 4-6 different sites collected and analyzed
for each GI section. While average values for the colon were comparable for the 3 monkeys, there was far more variability (up to 2
orders of magnitude) in values obtained from the small bowel segments, suggesting potential differences in viral clearance during ART.
In contrast, measuring gp120 via PET, an approximately 10-fold difference was observed between monkeys. b. These figures illustrate
the differences in qRT-PCR signals obtained from various collection sites from the same GI segment of a given animal on ART. It is
obvious from the data that the variation in viral RNA levels can range up to 2 orders of magnitude within the same tissue, especially for
the colon. This clearly has implications for the interpretation of data obtained from biopsies relative to an entire organ. All qRT-PCR
data is presented also in Supplementary Table 3.
Additional PET-CT results from an EC SIV-infected macaque.
a,b. Frontal, sagittal and axial views are presented, as well as magnified views (marked by a colored box and associated image
denoted by the same color outline) of regions of the frontal and sagittal sections. Single plane PET/CT cross-sections from macaque
EC 2 and 3, 36 hrs post injection of the contrast agent. EC 2 and 3 demonstrated PET signal foci in the GI tract, including the ileum,
jejunum, and colon, as well as within axillary, mesenteric, and inguinal lymph nodes, and male genital tract. IHC for p27 Gag results
from biopsy samples for each monkey are also presented. Infected mononuclear cells (black arrows) were detected in the rectum,
epididymis and jejunum, verifying the PET results. b. Cross-sections of frontal and axial views of macaque EC 3, an elite controller. For
the frontal views, each slice is 1.37 mm thick, 24 slices are displayed, amounting to a frontal view every 5.46 mm. The axial view
thickness is 5 mm and images are displayed every 3 mm. SIV positive signal is apparent throughout the GI tract, and within the lung.
Background signal, as expected, was high within the liver, heart, and kidneys.
Comparison of PET signal distribution in viremic and EC macaques.
Representative axial cross sections of chronically infected macaques (Viremic 2) and controller macaques (EC3). The uptake in the
chronically viremic infected animals (white arrows) tend to follow the length of the GI organs, while in the controllers (white arrows), the
signal localizes to specific foci, some of which are mesenteric lymph nodes (from CT), while in other cases they correspond to specific
regions of the small intestines or colon. The red arrows indicate uptake within the liver.
Additional supplementary information
Monkeys
Yerkes
designation
sex
age
years
Viremic 1
Viremic 2
Viremic 3
Viremic 4
Viremic 5
EC 1
EC 2
EC 3
EC 4
ART 1
ART 2
ART 3
ART 1
ART 2
ART 3
Control 1
Control 2
RFR‐11
RID‐9
RLC‐8
RCN‐10
RYY13
RUN‐10
RMP‐10
RBQ‐10
RQZ‐8
RUT‐13
RHY‐12
RQM‐11
RUT‐13
RHY‐12
RQM‐11
RHG‐7
RVE‐7
M
F
F
F
M
M
M
M
F
M
F
F
M
F
F
M
M
5
9
10
7
4
8
8
8
10
4
6
8
4
6
8
11
11
Body
Weight
kg
6.9
9.12
8.8
6
7.46
9.86
14.48
10.06
7.75
5.84
5.94
8.48
6.1
6
8.5
12.6
15.75
Dose
mCi
Time pi
Weeks
1.2
3.7
2.1
1
3
3
1.8
1.6
1.3
1
1
1
1
1
1
1.2
1.5
69
71
66
66
34
>6y
>6y
>6y
112
13
38
37
41
42
41
0
0
Mamu
alleles
A*001/B*008/B*017
‐/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
+/‐/‐
+/‐/‐
‐/‐/‐
‐/‐/‐
+/‐/‐
+/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
‐/‐/‐
SIV plasma
VL
mRNA/ml
1.65E+05
6.85E+05
2.89E+05
1.64E+06
7.53E+04
< 60
< 60
< 60
< 60
2.64E+06
3.39E+04
3.06E+04
< 60
< 60
< 60
< 60
< 60
Clinical
status
viremic
viremic
viremic
viremic
viremic
EC
EC
EC
EC
viremic *
viremic *
viremic *
ART
ART
ART
uninfected
uninfected
viremic * = pre‐ART
Supplementary Table 1. Macaques used in this study, including gender, weights,
probe specific activity, time post-infection with SIV and viral load at the time of imaging.
Supplementary Table 2. Kruskal-Wallis analysis of the statistical differences in
SUVmax measurements in individual organ systems, comparing animal status (infected
viremic vs uninfected, ECs vs infected viremic). The data analyzed here is depicted in
Figure 1 and 3. From this data it is clear that for all organ systems (yellow), the
differences in PET signals are statistically significant (p<0.05) except for the muscle.
For ECs vs viremic monkey comparisons, only the kidneys and axillary lymph nodes
(yellow) showed a statistical difference in the SUVmax. However, differences were
detected when using the SUVmean (p<0.05) (Figure 4) by the same statistical test.
a
Percent reduction in SUVmax after 4 weeks of ART
ART 1
ART 2
ART 3
AVERAGE
STDEV
ART 1
ART 2
ART 3
AVERAGE
STDEV
b
Mesenteric LN
Right Inguinal LN
Left Inguinal LN
Right Axil LN
Left Axil LN
Colon 1
Colon 2
Colon 3
Colon 4
Colon 5
Colon 6
Jejunum 1
Jejunum 2
Jejunum 3
Jejunum 4
Cecum
Ileum 1
Ileum 2
Ileum 3
Ileum 4
Spleen
Kidney R
Kidney L
Urethra
Testis
Epididymis
Saminal Gland
Prostate
Vagina
Ectocervix
Endocervix
Uterus
Ovary
Lung 1
Lung 2
Tonsil lingual
Tonsil pharyngeal
Nasal turbinate
Colon
Small bowel
64.36%
51.07%
46.81%
69.94%
50.71%
73.86%
53.96%
9.21%
64.96%
12.19%
NALT
43.67%
43.68%
70.11%
52.49%
15.26%
spleen testes
genital tract
27.61%
72.78%
37.33%
40.36%
NA
53.64%
43.81%
NA
71.10%
37.26%
8.53%
left inguinal LN
right inguinal LN left axillary LN
47.31%
NA
68.32%
68.27%
29.40%
58.02%
64.23%
81.12%
51.16%
59.94%
11.12%
55.26%
36.57%
59.16%
8.64%
72.78%
54.02%
16.89%
right axillary LN
68.37%
47.56%
60.70%
liver
29.55%
43.36%
51.41%
58.88%
10.52%
41.44%
11.06%
ART 1 (viral copies per ngRNA) ART 3 (viral copies per ngRNA) ART 2(viral copies per ngRNA)
2448.86
179.67
526.48
4104.47
1253.08
278.44
4440.32
353.65
1065.40
973.14
839.49
561.09
3749.70
325.57
602.91
60.42
68.32
14.47
36.61
6.54
15.15
10.32
7.92
41.11
73.07
49.65
40.38
77.01
37.30
21.49
34.56
36.63
335.37
0.21
Undetected
0.46
0.64
Undetected
0.48
12.67
Undetected
0.19
1.22
Undetected
0.31
4.83
1.66
7.73
6.75
0.49
356.88
0.91
Undetected
168.07
undetected
Undetected
25.18
0.96
Undetected
287.61
1824.07
64.07
58.09
2.53
0.06
0.17
23.81
0.18
0.41
3.64
No sample
No sample
0.25
No sample
No sample
0.36
No sample
No sample
2.62
No sample
No sample
0.79
No sample
No samples
No sample
0.92
Undetected
No sample
0.18
0.76
No sample
1.26
2.07
No sample
0.08
Undetected
No sample
0.01
Undetected
9.89
4.28
10.40
5.53
3.60
Undetected
77.01
Undetected
63.72
7.43
80.88
365.97
1.36
4.70
8.85
Supplementary Table 3. a. Percent reduction in SUVmax after 5 weeks of ART
treatment of chronically infected macaques. b. Tissue qRT-PCR results of 3 monkeys
chronically infected with SIV and sacrificed after 5 weeks of ART. At the time of
sacrifice, all three animals were aviremic, with less than 60 copies vRNA/ml plasma.
The data also illustrate the variability in the various samples and sites collected from the
colon, jejunum, cecum, ileum, and lung, illustrating the spatial heterogeneity of SIV
within organs from an animal. A portion of this data is plotted in Supplementary Fig. 6.
Supplementary Note 1. SIV localization in infected aviremic elite controller
macaques. Once the sensitivity and specificity of the immunoPET/CT method was
demonstrated, highlighted by its ability to distinguish uninfected from SIV infected ART
treated animals, the approach was used to image SIV infected elite controllers (EC).
ECs are individuals that maintain SIV or HIV replication levels to undetectable levels in
plasma for extended periods of time without ART1-3. The study of the pathogenesis,
viral persistence, and interventions in these individuals is challenging due to the
absence of detectable plasma viremia. We reasoned that EC monkeys would serve as
additional important candidates for an imaging technique that can identify potential sites
of residual virus replication. Therefore, four macaques identified as ECs were injected
with the 7D3 probe and imaged. In Figure 3, fused frontal, sagittal, and axial, PET/CT
images from 1 of the 4 LTNPs, EC 1, is displayed, focusing on the nasal associated
tissue, GI tract, inguinal/axillary lymph nodes and the genital tract. This animal had
been infected with SIV >6 years with viral loads of <60 copies of viral RNA per ml of
plasma for the last >5 years. The images of the controllers (Fig. 3 and Supplementary
Figure 7) show similarities with those of the viremic animals, but also marked
differences. They exhibited detectable uptake within the GI tract, genital tract
(epididymis), NALT, lungs, spleen and axillary lymph nodes, but the signal was
markedly more restricted to small foci within the positive organs. In addition to the
images displayed in Figure 3, images from EC 2 (Supplementary Fig. 7a) and individual
frontal and axial image slices throughout the entirety of EC 3 were studied and are
displayed (see Supplementary Fig. 7b) providing a more detailed view of the anatomical
localization of signals. The signal quantified using the SUVmax integration surprisingly
provided levels comparable to those seen in viremic animals (Figure 3b). However,
further analysis using the hierarchical ANOVA clearly showed that viremic animals were
statistically distinct from the ECs, with a p-value of 2.78e-5, and while each population
was distinct, individuals within viremic monkeys or ECs appeared homogenous (βj(i)=0;
p-value=0.9804). Individual organ differences were assessed by both Kruskal-Wallis
with statistical differences in the kidneys and axillary lymph nodes or by ANOVA (with
Bonferoni) where additional differences were detected (Supplementary Table 2). The
imaging results were verified via IHC (Figure 3c and Supplementary Fig. 7a,b) assays
performed on biopsies of the rectum, jejunum, and the epididymis. SIVgag+ cellular
infiltrates (black arrows) were detected in each of these tissues with the highest
prevalence within jejunum and rectum, corroborating the PET imaging results. These
animals were not sacrificed because they are part of a number of ongoing studies
aimed at unraveling mechanism(s) by which these SIV infected animals maintain their
EC status.
To further clarify and quantify the differences between ECs and viremic animals,
the SUVmean was measured specifically within the GI tract (Figure 4a). In addition, the
voxel fractions (fraction of total volume of GI tract) that contained SUVs above 1 and
less than 3.3 (excludes interfering signals) were compared (Fig. 4a). Using the mean
signal as a yardstick, it was found that the GI tract from the chronic SIV+ viremic
macaques exhibited 2.1 times the signal obtained on the ECs. Moreover, when the
volume of tissue with uptake between 1 and 3.3 was examined, the chronic SIV+
macaques exhibited 6.38 times the signal of the ECs. These data clearly demonstrate
that while the viremic SIV+ macaques and the ECs both contain regions of high uptake
with similar maximal intensity, the signal distribution is markedly different. In order to
more quantitatively compare the variation in the signal distribution within regions of the
GI tract, two Haralick texture features4, the angular second moment and contrast were
calculated for the same size ROI (Fig. 4b,c,d) within both chronically infected and elite
controller animals. The angular second moment is a measure of homogeneity, while the
contrast metric represents the local variations within an image or ROI. These metrics
are inversely proportional to each other, and therefore ideal to compare the distributions
of signal within the GI tract of SIV infected viremic animals and ECs. In Figure 4b, both
metrics were plotted; the angular second moment for the chronically infected animals
was 4.6X higher than in the ECs, while the contrast in the ECs was 8.4X higher than in
the chronically viremic animals. These objective metrics clearly demonstrate that the
signal within the GI tract of the SIV infected viremic animals is more homogeneous than
the signal within the GI tract of the ECs, reflecting the conclusion that in ECs, the signal
is located within foci or small regions. (Figure 3 and 4c,d).
References
1.
Saez-Cirion A, Bacchus C, Hocqueloux L, Avettand-Fenoel V, Girault I, Lecuroux
C, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the
interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS
Pathog. 2013; 9(3): e1003211.
2.
Grabar S, Selinger-Leneman H, Abgrall S, Pialoux G, Weiss L, Costagliola D.
Prevalence and comparative characteristics of long-term nonprogressors and HIV
controller patients in the French Hospital Database on HIV. AIDS. 2009; 23(9): 1163-9.
3.
Zaunders J, van Bockel D. Innate and Adaptive Immunity in Long-Term NonProgression in HIV Disease. Front Immunol. 2013; 4: 95.
4.
Haralick RM, Shanmugam K, Dinstein I. Textural Features for Image
Classification. IEEE Transactions on Systems, Man, and Cybernetics. 1973; 3(6): 61021.

Whole-body immunoPet reveals active siV dynamics in viremic and

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