JOURNAL OF VIROLOGY, Apr. 2008, p. 3713–3724
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 7
Simian Immunodeficiency Virus SIVagm Dynamics in African
Ivona Pandrea,1,2* Ruy M. Ribeiro,3Rajeev Gautam,4Thaidra Gaufin,4Melissa Pattison,4
Mary Barnes,4Christopher Monjure,4Crystal Stoulig,1Jason Dufour,5Wayne Cyprian,5
Guido Silvestri,6Michael D. Miller,7Alan S. Perelson,3and Cristian Apetrei4,8
Divisions of Comparative Pathology,1Microbiology,4and Veterinary Medicine,5Tulane National Primate Research Center,
Covington, Louisiana 70433; Department of Pathology, School of Medicine, Tulane University, New Orleans,
Louisiana 701122; Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos,
New Mexico 875453; Department of Pathology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 191076; Gilead Sciences, Inc., Foster City, California 944047; and
Department of Tropical Medicine, School of Public Health,
Tulane University, New Orleans, Louisiana 701128
Received 7 November 2007/Accepted 15 January 2008
The mechanisms underlying the lack of disease progression in natural simian immunodeficiency virus (SIV)
hosts are still poorly understood. To test the hypothesis that SIV-infected African green monkeys (AGMs)
avoid AIDS due to virus replication occurring in long-lived infected cells, we infected six animals with SIVagm
and treated them with potent antiretroviral therapy [ART; 9-R-(2-phosphonomethoxypropyl) adenine (teno-
fovir) and beta-2,3-dideoxy-3-thia-5-fluorocytidine (emtricitabine)]. All AGMs showed a rapid decay of plasma
viremia that became undetectable 36 h after ART initiation. A significant decrease of viral load was observed
in peripheral blood mononuclear cells and intestine. Mathematical modeling of viremia decay post-ART
indicates a half-life of productively infected cells ranging from 4 to 9.5 h, i.e., faster than previously reported
for human immunodeficiency virus and SIV. ART induced a slight but significant increase in peripheral CD4?
T-cell counts but no significant changes in CD4?T-cell levels in lymph nodes and intestine. Similarly, ART did
not significantly change the levels of cell proliferation, activation, and apoptosis, already low in AGMs
chronically infected with SIVagm. Collectively, these results indicate that, in SIVagm-infected AGMs, the bulk
of virus replication is sustained by short-lived cells; therefore, differences in disease outcome between SIVmac
infection of macaques and SIVagm infection of AGMs are unlikely due to intrinsic differences in the in vivo
cytopathicities between the two viruses.
One of the most intriguing pathogenic features of simian
immunodeficiency virus (SIV) infection is that African nonhu-
man primate (NHP) natural hosts, such as African green mon-
keys (AGMs), sooty mangabeys (SMs), mandrills, and chim-
panzees, naturally or experimentally infected with their
species-specific SIV generally do not progress to AIDS (3, 5,
16, 43, 50, 59, 65, 66). This feature is in striking contrast to
pathogenic lentiviral infections of humans and macaques, for
which the outcome of infection is disease progression (32). It is
now widely acknowledged that a better understanding of the
mechanisms of the lack of disease progression in natural SIV
infections may be needed for understanding the pathogenesis
of AIDS in human immunodeficiency virus (HIV)-infected
individuals (70, 71).
Both HIV type 1 (HIV-1) and HIV-2 originated from cross-
species transmission events of SIVs naturally infecting chim-
panzees/gorillas and SMs, respectively (73). SIVsmm/SIVmac
strains that are pathogenic in rhesus macaques (RMs) also
originated from naturally infected SMs (2). Infection of SMs or
RMs and AGMs or pigtailed macaques with the same strains
of SIVsmm/SIVmac or SIVagm, respectively, results in differ-
ent outcomes, with disease progression in macaques and per-
sistent nonprogressive infection in natural African NHP hosts
(17, 25, 29, 65). Therefore, the differences in pathogenic po-
tentials do not appear to be virus related. Importantly, some of
the immunological consequences of SIV infection shared by
pathogenic and natural SIV infections are very similar, most
notably the early and massive CD4?T-cell depletion at the
mucosal sites (19, 33, 38, 48). Hence, the current view is that
the main reason behind the lack of disease progression in
natural African hosts lies in a better adaptation of the host in
response to the highly replicating virus rather than it reflecting
an infection with less pathogenic viral strains. This improved
adaptation of the host immune system does not mean stronger
or broader immune responses to viral antigens (10, 24, 76; I.
Pandrea, unpublished data). Moreover, studies by us and oth-
ers have shown that normal levels of immune activation, T-cell
proliferation, and apoptosis are characteristic for the chronic
phase of SIV infection in natural hosts (7, 29, 43, 44, 46, 50, 65,
66) and, at least in the case of SIVagm infection of AGMs, may
be due to an anti-inflammatory response very rapidly estab-
lished upon SIVagm infection (30). These responses are dif-
ferent from pathogenic infections in humans and macaques,
which are characterized by significant increases in immune
activation, the levels of which have been reported to be pre-
dictive for disease progression (13, 68). Furthermore, in patho-
* Corresponding author. Mailing address: Division of Comparative
Pathology, Tulane National Primate Research Center, 18703 Three
Rivers Road, Covington, LA 70433. Phone: (985) 871-6408. Fax: (985)
871-6510. E-mail: firstname.lastname@example.org.
?Published ahead of print on 23 January 2008.
genic infections, immune cell proliferation and apoptosis are
severely compromised (42, 56).
One of the hypotheses proposed to explain the lack of dis-
ease progression in natural African NHP hosts is that the
better preservation of peripheral CD4?T cells and the partial
immune restoration of mucosal CD4?T cells in the presence
of high levels of viral replication may be due to a different in
vivo viral cythopathicity (64), the corollary of this being a
significantly longer average life span of infected cells. The in
vivo life span of infected cells has been previously measured in
pathogenic HIV/SIV infections using potent antiretroviral
therapy (ART) (27, 37, 54, 55, 77, 79). These studies reported
a two-phase decline in plasma viral load (VL) after the admin-
istration of ART: an initial rapid decline of viremia, due to loss
of short-lived virus-producing cells (activated CD4?T cells),
followed by a slower decline, occurring as a consequence of
loss of longer-lived virus-producing cells (resting T cells or
macrophages) (53, 55). Mathematical modeling showed that
the bulk of HIV replication (93 to 99%) occurs in recently
infected cells that die soon after infection, with an average life
span calculated on the order of 1 day after the start of viral
production, and only 1 to 7% of virus production derives from
long-lived cells, which have an average life span on the order of
1 to 4 weeks (53).
In this study, we followed a similar approach and estimated,
from the kinetics of viral decline in vivo, the life span of
virus-producing cells in SIVagm-infected AGMs receiving
ART during chronic infection. We report that the bulk of
SIVagm replication in vivo is sustained by short-lived infected
cells. The decay of viremia following ART was more rapid in
SIVagm.sab-infected AGMs than in HIV-infected individuals
and SIV-infected RMs. These data suggest that the lack of
disease progression in SIVagm-infected AGMs is unlikely to
be related to reduced intrinsic virus cytopathicity, and the data
suggest a key role for species-specific host factors in determin-
ing the outcome of a primate lentiviral infection.
MATERIALS AND METHODS
Animals. Six Caribbean AGMs (Chlorocebus sabaeus), originating from St.
Kitts Island, were included in this study. The animals were adults (mean age, 7
years). All animals were negative for simian T-cell lymphotropic virus (Vironos-
tika human T-cell lymphotropic virus types I and II enzyme-linked immunosor-
bent assay; BioMerieux, Durham, NC) and SIV by an in-house SIVagm.sab-
specific enzyme-linked immunosorbent assay and were housed at the Tulane
National Primate Research Center, an AAALAC International-accredited facil-
ity. Housing and handling of animals were in accordance with the Guide for the
Care and Use of Laboratory Animals (40) and the Animal Welfare Act. All
protocols and procedures for the animal studies were reviewed and approved by
the Tulane University Institutional Animal Care and Use Committee.
SIVagm.sab infection. To avoid selection of viral variants in vitro, inocula used
inthis study consistedof plasma
SIVagm.sab92018-infected AGM. Plasma titers were determined on SupT1 as
described elsewhere (9), and all six AGMs were inoculated with plasma contain-
ing the equivalent of 300 50% tissue culture infective doses of SIVagm.sab92018.
Antiretroviral therapy. All AGMs included in this study were treated with
nucleotide reverse transcriptase inhibitors (NRTIs) 9-R-(2-phosphonome-
thoxypropyl) adenine (PMPA; tenofovir) and beta-2,3-dideoxy-3-thia-5-fluoro-
cytidine (FTC; emtricitabine) for 21 days. Antiretroviral (ARV) drugs were
administered starting from day 254 post-SIV inoculation, when levels of plasma
viremia had reached the set point and were remarkably stable. Subcutaneous
injections of both drugs were given at doses of 30 mg/kg of body weight/day. Prior
to treatment, monkeys were trained for subcutaneous injections, which allowed
drugs to be administered without repeatedly undergoing anesthesia. PMPA and
FTC were kindly provided by Gilead (Foster City, CA).
Blood and tissue collection. In order to model the dynamics of virus replica-
tion, thorough sampling schedules during the primary SIVagm.sab infection and
antiretroviral treatment were designed. Blood (4.9 ml, EDTA anticoagulated)
was collected from the femoral vein at days ?7 and 0, 3, 6, 8, 10, 13, 15, 18, 20,
28, 30, 42, 56, 72, 100, 132, 185 post-SIVagm.sab inoculation. Moreover, every 7
days between day 100 and day 132, 4.9 ml of blood was collected (with EDTA)
to determine the weekly variation of VLs in SIVagm.sab-infected AGMs. Then,
the animals were not sampled for 4 months to minimize stress prior to treatment.
ART was initiated at 254 days post-SIV inoculation. During the ART, the
sampling schedule was every 2 hours during the first 6 hours, every 6 hours during
the first 2 days, and then every 2 days during the first 2 weeks and every 3 days
during the third week. After the treatment interruption, samples were collected
every day for 4 days, then every 3 days for 2 weeks, and weekly for another 2
weeks. A final sample was collected 7 weeks after treatment ceased.
Whole blood was used for flow cytometry within 1 h of collection. Plasma was
separated within 2 h of collection and stored in aliquots at ?80°C until used for
Endoscopically guided pinch biopsies from the proximal jejunum, intestinal
resections of the small intestine, and excisional biopsies of axillary and inguinal
lymph nodes (LNs) were collected from all the animals as previously described
(46, 48). LNs and intestine were sampled before infection (day ?14) and at
different time points during the acute (days 10, 21, and 28 postinfection [p.i.])
and chronic (days 42, 72, 100, 132, 200, and 230 p.i.) phases of SIVagm infection.
Intestinal biopsies and LN biopsies were also performed at the initiation, at the
end, and at 72 days post-antiretroviral treatment.
Isolation of lymphocytes. Mononuclear cells were separated from the blood
through Ficoll density gradient centrifugation. Lymphocytes from the intestine
and LNs were isolated and stained for flow cytometry as previously described (46,
48). Briefly, lymphocytes were isolated from intestinal biopsies using EDTA
followed by collagenase digestion and Percoll density gradient centrifugation
(48). Lymphocytes were isolated from the axillary LNs by gently mincing and
pressing tissues through nylon mesh screens.
Antibody detection. Anti-SIVagm.sab92018 antibody dynamics were moni-
tored by using a SIVagm.sab-specific enzyme immunoassay (PIV-EIA) based on
peptides mapping the conserved Gp41 immunodominant region and the highly
variable V3 loop of SIVagm.sab2 (67). Serological reactivity was confirmed by
Western blotting for all the monkeys included in this study (Zeptometrix Corp.,
Viral RNA quantification. Viral RNA was extracted from 540 ?l of plasma
using the QIAamp viral RNA extraction kit (Qiagen, Valencia, CA). During the
ART, in order to improve the efficacy of testing, viral RNA was extracted from
the maximum amount of plasma available (840 to 1,000 ?l). RNA was also
extracted from 3 ? 106mononuclear cells isolated from blood, LNs, and intes-
tinal biopsies using an RNeasy kit (Qiagen, Valencia, CA). A DNase digestion
step was applied to the tissue extractions. Viral RNA was extracted from pe-
ripheral blood mononuclear cells (PBMCs), LNs, and intestine prior to, at the
end of, and at 72 days post-antiretroviral treatment.
VL quantification was done by real-time PCR as previously described (30, 49).
Briefly, total RNA was retrotranscribed into cDNA using the TaqMan Gold
reverse transcription-PCR kit and random hexamers (PE, Applied Biosystems,
Foster City, CA). PCRs were carried out in a spectrofluorometric thermal cycler
(ABI Prism 7700; PE). Quantification was based on the amplification of 180 bp
located in the long terminal repeat (LTR) region. This region is very conserved
within different SIVagm strains. The SIVagm.sab primers and probe were the
following: J15S (5?-CTG GGT GTT CTC TGG TAA G-3?), 5? J15S (5?-CAA
GAC TTT ATT GAG GCA AT-3?), and J15P (6-carboxyfluorescein–CGA ACA
CCC AGG CTC AAG CTG G–6-carboxytetramethylrhodamine) as previously
described (9). SIVagm.sab cDNA was added to the universal master mix (PE,
Applied Biosystems) containing a 10 ?M concentration of each primer and 10
?M concentration of the probe. All PCRs were carried out in duplicate in
parallel with a negative non-RT control reaction. The PCR cycling conditions
were as follows: a first cycle of denaturation (95°C, 10 min), followed by 45 cycles
of denaturation (95°C, 10 s), annealing (50°C, 30 s), and extension (72°C, 30 s).
Absolute viral RNA copy numbers were deduced by comparing the relative
signal strength to corresponding values obtained for seven 10-fold dilutions of
standard RNA, which were reverse transcribed and amplified in parallel. The
RNA standard consisted of a larger LTR region of SIVagm.sab92018 that was
PCR amplified with primers LTR2A (5?-AAC TAA GGC AAG ACT TTA TTG
AGG-3?) and LTR4S (5?-ACT GGG CGG TAC TGG GAG TGG CTT-3?). The
PCR product was cloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA). In
vitro transcription was then performed using the MEGAscript kit (Ambion,
Austin, TX). Known amounts of the SIVagm LTR standard RNA were used to
3714PANDREA ET AL. J. VIROL.
determine the target copy numbers. The detection limit of the SIVagm quanti-
fication assays was 100 RNA copies/ml of plasma.
Viral RNA quantification in tissues. Viral RNA was extracted from 5 ? 105to
106cells from intestine and LNs with RNeasy (Qiagen, Valencia, CA), and VLs
were quantified as described elsewhere (46). Simultaneous quantification of 18S
rRNA (rRNA control reagent kit; Perkin-Elmer) was used to normalize the
RNA input from cells (21). The assay sensitivity was 100 RNA copies/105cells.
Flow cytometry. Mononuclear cells derived from peripheral blood, intestinal
biopsies, and LNs were stained for flow cytometric analysis using four-color
staining combinations with the following monoclonal antibodies: CD3-fluo-
rescein isothiocyanate (FITC), CD20-phycoerythrin (PE), CD8-peridinin
chlorophyll A protein (PerCP), CD4-allophycocyanin (APC), CCR5-PE, HLA-
DR-PerCP, CD95-FITC, CD28-APC, CD69-APC or CD69-FITC, CD25-PE, Ki-
67-FITC, and Annexin V–PE–7-amino actinomycin D (7AAD; BD Biosciences
Pharmingen, San Diego, CA). Cells were incubated with an excess amount of
monoclonal antibodies at 4°C for 30 min, followed by a phosphate-buffered saline
wash (400 ? g; 7 min) and fixation in 2% paraformaldehyde. Whole blood was
stained using a whole blood lysis technique previously described (46). Samples
were stained for Ki-67 and apoptosis using a Ki-67 R-PE-conjugated mouse
anti-human monoclonal antibody set and the Annexin V-PE apoptosis detection
kit I (BD Pharmingen) as per the manufacturer’s instructions. Apoptotic CD4?
T cells were defined as Annexin V?7AAD?, whereas the necrotic CD4?T
cells were defined as Annexin V?7AAD?. Stained cells were analyzed with a
FACSCalibur flow cytometer (BD Immunocytometry Systems) and analyzed
with CellQuest software (BD). CD4?and CD8?T-cell percentages were ob-
tained by first gating on lymphocytes and then on CD3?T cells. Memory,
activation, proliferation, and apoptosis markers were determined by gating on
lymphocytes, then on CD3?T cells, and finally on CD4?CD3?or CD8?CD3?
IHC staining and ISH. Immunohistochemical (IHC) staining and in situ hy-
bridization (ISH) were performed on formalin-fixed, paraffin-embedded tissues,
as described elsewhere (48). SIVagm was detected in tissues by ISH. Sections
were subjected to high-temperature unmasking, treated with 0.2 N HCl, and
hybridized overnight at 45°C with either sense or antisense SIVagm digoxigenin-
UTP-labeled riboprobe, blocked with normal sheep serum, incubated with sheep
antidigoxigenin-alkaline phosphatase, and incubated with the HNPP fluores-
cence detection set (Roche). The SIVagm-infected cell phenotype was deter-
mined after ISH by incubating sections with rabbit anti-human CD3 (DAKO,
Carpinteria, CA) or mouse anti-human macrophage (HAM56; DAKO), fol-
lowed by the appropriate goat anti-mouse or goat anti-rabbit antibodies labeled
with Alexa 488 (ABC method; Vectastain Elite ABC kit). Negative controls
included an antisense probe with uninfected tissues, a sense probe with infected
tissues, an antisense probe with infected tissues, and anti-rabbit or anti-mouse
secondary antibodies only.
Mathematical modeling of data. For the dynamic analysis of our data, we used
a simplification of the standard model of viral dynamics (41, 52). The model
describes the changes in time of the following populations: target cells (T),
infected cells (I), and virus (V). Uninfected cells are produced at the total rate ?
and die at the per capita rate d. In addition, they can become infected at a rate
proportional to the viral load ?V, generating infected cells, which are lost at the
rate ?, higher than d. Virions are produced from infected cells at a rate of p per
cell and cleared by all mechanisms at the rate c (52). Thus, we obtain the
dt? ? ? dT ? ?VT,
dt? ?VT ? ?I, and (1)
dt? pI ? cV
When a monkey is first infected, the virus will grow exponentially as V(t) ap-
proaches ?ert, where r is the rate of growth, which can be determined from the
slope of the increase of virus in a plot of log Vtversus time. Moreover, it can be
shown analytically (60) that this initial growth rate (r) is related to the basic
reproduction number of the virus R0, such that
where the last approximation is valid when c is ? ?r. The basic reproduction
number is a fundamental quantity, because it expresses the condition for the
virus to infect a host: when R0is ?1, the virus can spread and cause chronic
infection, but if R0is ?1, the virus cannot cause infection (41).
We assumed that the set point VL (V0) is reached after 4 to 6 weeks of
infection. ART was initiated at day 254 p.i. Assuming that this treatment is 100%
efficient, as it was assumed before (77), the solution of the system (from equation
group 1) is:
V?t? ? V0
c ? ?
This equation indicates that the behavior of virus under therapy should follow a
double exponential decay pattern. However, the data (see Fig. 1 and Results,
below) indicate that the decay follows a single exponential pattern. This result is
not surprising, because from other models of infection (such as HIV or SIV in
RMs), we know that c is much larger than ?. When this happens, expression
(from equation 3) can be approximated by a term containing only the exponen-
tial in ?, such that
V?t? ? V0
c ? ?e??t
Again, on a logarithmic scale this represents a simple linear decay, as observed.
In addition, the slope of that decay corresponds to the infected cell loss rate, ?.
Statistical analyses. To estimate both the rate of initial viral expansion upon
infection and the infected cell loss rate, we fitted a linear regression to the VL
data, using a mixed effects model approach (57). In this method, all the monkeys
were fitted simultaneously and the best population estimate for the slopes was
obtained. We also estimated rates of virus decline postpeak in acute infection
and viral rebound after treatment cessation by using mixed effects models.
Results are presented as means ? standard deviations, and P values were con-
sidered significant if they were less than 0.05.
Dynamics of SIVagm.sab replication in experimentally in-
fected AGMs. We measured and modeled quantitatively the
dynamics of SIVagm.sab replication in AGMs during the acute
phase, the attainment of the viral quasi-steady state (the set
point), and while undergoing treatment.
Primary infection. VLs increased exponentially, reaching a
peak around day 8 for all monkeys, with a geometric mean of
3.2 ? 107copies/ml. The average slope of increase was 0.68 ?
0.06 log10day?1, corresponding to a doubling time in VL of
about 11 h. The VL then decreased with a half-life of about 4.5
days, reaching a quasi-steady state of 1.2 ? 105copies/ml by
days 28 to 42 p.i. This pattern of viral replication was very
similar to that of pathogenic SIVmac infections (26, 38, 48).
The steady-state VL was remarkably stable in all animals, with
an average coefficient of variation, within individual animals, of
only 5% between days 42 and 254, the time of initiation of
ART. To confirm that VLs were constant during the steady
state, we monitored the weekly variation in VLs between days
100 and 132 p.i. As shown in Fig. 1a, no significant variations
in VLs could be observed during this interval.
Combined ISH for SIVagm RNA and IHC for either mac-
rophages or CD3?T cells confirmed the high viral replication
and showed that SIVagm colocalizes with T cells (Fig. 2b) and
not macrophages (Fig. 2a), suggesting that SIVagm preferen-
tially infects T cells during acute infection.
Antiretroviral treatment. Figure 1 shows the data obtained
after treating the six AGMs with daily PMPA plus FTC (see
Materials and Methods). After a short delay, possibly due to
drug pharmacokinetics, the VL decays very rapidly. Figure 1c
shows that there is a clear rebound of virus, starting at about
18 h posttreatment. This rebound is presumably due to the
pharmacokinetic elimination of the drugs (20, 75) and is not
VOL. 82, 2008SIVagm DYNAMICS IN AFRICAN GREEN MONKEYS3715
commonly seen in the setting of HIV/SIV treatment, although
the frequency of measurement early posttherapy in our study is
higher than other typical macaque studies (18, 41). Neverthe-
less, starting at 36 h, all monkeys showed a continued decay of
virus that eventually fell below the limit of detection (100
The observed rebounds make it difficult to fit the “standard”
model of treatment to the data (equation 3 in Materials and
Methods). Nevertheless, the overall decay observed clearly
indicates an effect of the drug. Moreover, this decay appears
essentially linear on the log scale, once we discount the puta-
tive pharmacokinetic rebound. In this case, we can fit an ap-
proximation of the standard model, which assumes that the
clearance of free virus is much faster than the loss of infected
cells, as is well known in both HIV and SIV pathogenic infec-
tions (27, 41, 55, 58, 77). From equation 4, we then expect a
linear decay, with the slope given by the loss rate of infected
cells. We have tried two different strategies to fit the data using
simple linear regression: (i) strategy a, using only daily data
(i.e., time zero and 1, 2, 3, 4, 5, and 6 days); and (ii) strategy b,
fitting only the fastest decay observed on the first day. This
analysis was done using a linear mixed effects approach, as
described in Materials and Methods.
The average decay rates and their corresponding half-lives
were as follows: for strategy a, decay ? ?0.76 log10/day and t1/2?
9.5 h; for strategy b, decay ? ?1.97 log10/day and t1/2? 3.7 h.
These decays are very fast, even if they are minimal estimates,
since we assumed 100% efficacious therapy. In fact, these de-
cays are faster than previously reported for HIV in humans
(37) and SIV in RMs (41) or cynomolgus macaques (4). They
are also faster than those calculated for SIVsmm infection of
SMs (18). One contributing factor for the faster virus and cell
turnovers in AGMs may be that ART is more effective during
SIVagm infection for reasons of bio-distribution or the higher
After the discontinuation of ART, the virus rebounded to
levels comparable to those prior to the initiation of treatment.
It is interesting that the virus remained undetectable for up to
13 days in some monkeys before showing an exponential re-
bound, with an average doubling time of 27 h, which is much
slower than that observed during primary infection. This
slower rebound compared to primary infection might be due to
the action of immune responses or to a lower availability of
target cells, since during the chronic infection the levels of
mucosal CD4?T cells are never restored to the baseline levels
in natural African NHP hosts of SIV, as we have previously
reported (19, 48).
SIV RNA quantification in PBMCs, LNs, and intestine in-
dicated that this short-term ART had only a limited impact on
viral burden. The reduction in tissue RNA loads was more
prominent in PBMCs and intestine (Fig. 1d and f, respectively)
than in the LNs (Fig. 1e).
These data indicate that natural SIVagm infection of AGMs
is similar to HIV infection of humans in that suppression of de
novo infections by ART results in a rapid and profound decline
of plasma viremia, thus suggesting that the bulk of virus rep-
lication occurs in short-lived infected cells.
Calculation of the basic reproduction number, R0. The num-
ber of infected cells resulting from one infected cell introduced
in a totally uninfected population is the basic reproduction
number, R0, of the infection. This quantity is important be-
cause it has a threshold behavior: when R0is smaller than 1,
the infection will extinguish itself, since it generates less than
one infected cell per initial infected cell; if R0is ?1, then the
infection will spread. From the data collected in this study,
including the initial growth rate of the infection and the loss
rate of infected cells, we can estimate R0for SIVagm in African
green monkeys (see Materials and Methods). One unknown,
however, is the time interval between cells being infected and
starting to produce virus, the intracellular delay. This delay will
affect the estimate of R0(36, 41, 60) when this number is
calculated based on the exponential rate of viral growth during
early infection: longer delays result in larger R0values. Thus,
we estimated R0for a fixed intracellular delay of 1 day, as done
before (35, 41), and obtained an R0of 6.3 ? 1.3 (harmonic
mean). This relatively small value for R0is consistent with the
fast loss rate of cells estimated above.
Dynamics of CD4?T-cell counts during SIVagm infection.
In pathogenic HIV and SIVmac infections of humans and
RMs, there is a progressive decline of CD4?T cells (12). In
patients under highly active ART (HAART), the control of
viral replication results in increases in CD4?T-cell counts and
significant decreases in the fraction of activated and prolifer-
ating T cells, which are associated in untreated patients with
a progressive decline of circulating CD4?T cells (23, 31, 39,
FIG. 1. (a) Dynamics of SIVagm replication in AGMs during follow-up. (b) During acute infection, a very active replication, with a pattern that
is similar to that of other pathogenic or nonpathogenic infections, was observed. The set point was established between days 28 and 42
postinfection. (c) The administration of antiretroviral treatment induced a rapid reduction in viral loads, followed by a short rebound. This initial
effect was followed by a complete control of VLs starting from 42 h. (d to f) Antiretroviral treatment significantly reduced SIVagm RNA VLs in
PBMCs (d) and intestine (f) but had a limited impact on RNA VLs in the lymph nodes (e), as illustrated with SIVagm RNA VL quantification
before treatment, at the end of treatment, and 7 weeks later.
FIG. 2. Combined in situ hybridization for SIV and immunohisto-
chemistry for either macrophage (HAM56) (a) or lymphocyte (CD3)
(b) markers demonstrates that during the primary infection, the ma-
jority of the SIVagm-infected cells are lymphocytes.
VOL. 82, 2008SIVagm DYNAMICS IN AFRICAN GREEN MONKEYS3717
Primary infection. During acute infection, similar to patho-
genic SIVmac infection (38), the initial increase in virus counts
was accompanied by a significant loss of CD4?T cells in the
periphery, with counts reaching a nadir between days 6 and 10
and an average depletion of 46% of peripheral CD4?T cells
(an average slope of ?15 cells/?l/day between challenge and
the nadir; P ? 0.002). However, similar to pathogenic infec-
tion, the absolute number of CD4?T cells then showed a
partial recovery from day 42 onwards (until the start of treat-
ment on day 254), with the new CD4?T-cell steady-state level
approximately 25% lower than preinfection levels (Fig. 3a).
Tissue CD4?T-cell dynamics during the acute infection
showed patterns similar to those previously reported by our
group (46, 48).
Antiretroviral treatment. Over the first 14 days of treatment,
the CD4?T-cell count increased slightly, with an average slope
of ?4 cells/?l/day (P ? 0.027) (Fig. 3b). This increase seems to
be driven mostly by an increase in naı ¨ve CD28?CD4?T cells
of ?4.6 cells/?l/day (P ? 0.0003) over the first 10 days of
treatment. As mentioned above, after treatment discontinua-
tion, the virus remained undetectable for up to 2 weeks. When
the virus rebounded, there was an overall decay in CD4?T-cell
numbers in the blood with an average slope of ?2.2 cells/?l/
day (Fig. 3b). This decay was significant (P ? 0.0013) despite
the considerable variability of T-cell numbers. Again, naı ¨ve T
cells contributed to this decay, with an average slope of ?0.97
cells/?l/day (P ? 0.0013) after viral rebound.
During the short-term ART, no significant change in the
CD4?T cell numbers was observed in either LNs (data not
shown) or gut-associated lymphoid tissue (data not shown).
Minor fluctuations in the percentages of memory (CD28?
CD95?) and effector (CD28?CD95?) CD4?T cells were
observed during the ART administration (data not shown).
The lack of a significant increase in the fraction of these CD4?
T-cell subsets in ART-treated SIVagm-infected AGMs was not
entirely unexpected, given that these animals had relatively
high baseline levels of CD4?T cells. In the same way, stopping
therapy did not result in a significant decrease in the percent-
ages or numbers for CD4?T-cell subsets (data not shown),
confirming that in natural SIV infection significant CD4?de-
pletion only occurs in the presence of high levels of viral
replication and that maintenance of CD4?T-cell homeostasis
during steady-state viral replication may be related to CD4?
T-cell recovery (48).
Dynamics of T-cell turnover, immune activation, and apop-
tosis during ART. In HIV-infected individuals, suppression of
virus replication by HAART results in a rapid decrease in
T-cell turnover and reduced T-cell activation (22, 23, 34, 39).
To investigate the impact of viral replication on the level of
T-cell activation, we longitudinally assessed the expression of
the immune activation markers DR, CD69, and CD25 on T
cells. As previously reported (46, 48), during acute infection,
both CD4?and CD8?T cells showed transient increases in the
levels of immune activation (data not shown) However, during
ART administration and posttreatment virus rebound, no sig-
nificant dynamics of immune activation were observed in
SIVagm-infected AGMs, as illustrated by the percentage of
DR?CD4?T cells which remained more or less constant
throughout treatment and virus rebound (Fig. 4a). In contrast,
the percentage of DR?CD8?T cells increased during the
virus rebound (Fig. 4b), but this increase was not statistically
significant (P ? 0.09). No significant changes in DR?CD4?T
cells or DR?CD8?T cells were observed in the LNs or
intestine (data not shown), probably because the treatment was
administered only for a short time interval and did not signif-
icantly impact the tissue VL dynamics (Fig. 1d to f). The
peripheral blood and tissue dynamics of other immune activa-
tion markers on the CD4?and CD8?T cells (CD25 and
CD69) were very similar to those of DR?CD4?and DR?
CD8?T cells (data not shown), showing that short-term sup-
pression of viral replication induces only minor changes in the
FIG. 3. (a) Longitudinal flow cytometric analysis of absolute counts of peripheral CD4?T cells in SIVagm-infected AGMs showed a transient
depletion of CD4?T cells during acute infection, followed by rebound of CD4?T cells to near preinfection values and very good preservation
during the follow-up. (b) During antiretroviral treatment, a slight but significant increase in CD4?T-cell counts was observed. After treatment,
virus rebound induced a slight but significant depletion of CD4?T cells.
3718PANDREA ET AL.J. VIROL.
level of immune activation in AGMs naturally infected with
To assess ART-induced changes in T-cell turnover, we mea-
sured the expression of the proliferation marker Ki-67. As we
previously reported (48), a transient increase in Ki-67 expres-
sion by CD4?T cells occurs during acute SIVagm infection of
AGMs (data not shown), followed by a return to baseline levels
during steady-state infection. The initiation of ART did not
induce any significant reduction in the fraction of proliferating
CD4?T cells of SIVagm-infected AGMs. Interruption of ther-
apy was associated with an increase in the percentage and
numbers of proliferating CD4?T cells that reached levels
higher than those observed prior to treatment in five of six
animals (Fig. 4c). These data were quite variable for each
FIG. 4. Dynamics of immune activation (DR), cell proliferation (Ki-67), and apoptosis (Annexin V) markers during antiretroviral treatment
of SIVagm-infected AGMs. No significant changes in the dynamics of CD4?-DR?(a) or CD8?-DR?(b) cells were observed. A significant increase
in proliferation for both CD4?(c) and CD8?(d) cells was observed that was simultaneous with the posttreatment virus rebound. No impact on
the dynamics of apoptotic cells in the intestine was observed (e).
VOL. 82, 2008 SIVagm DYNAMICS IN AFRICAN GREEN MONKEYS3719
monkey over time, and we smoothed the data by calculating a
moving average. As seen in Fig. 4c, there was a clear increase
in the percentage of Ki-67?CD4?T cells in all but one (FV76)
of the AGMs, starting from the end of treatment for 2 or 3
weeks. This slight increase of ?0.06% per day was highly
significant (P ? 0.0001), even when the outlier (FV76) was
included in the analysis (P ? 0.002). The analysis of Ki-67?
CD4?T-cell counts showed a trend for increases in these cells
from the start of treatment to approximately day 43 post-
initiation of treatment, when the virus had rebounded back to
pretreatment levels. This increase, with an average across the
monkeys of 0.08 cells/?l/day, was significant (P ? 0.024) (Fig.
4c). Interestingly, the percentage of CD4?T cells expressing
Ki-67 in the LNs also tended to increase from the end of
therapy to day 51, after the viral rebound (P ? 0.062) (data not
No significant changes in the levels of proliferating CD8?T
cells were observed, except in one AGM (FV71), during ART
or post-treatment interruption (Fig. 4d). A slight transient
increase in CD8?T-cell proliferation occurred at the time of
treatment interruption, likely as a response to the increased
VLs. Ki-67?CD8?T-cell levels returned to pretreatment lev-
els when viral replication reached the preinfection set point
The dynamics of apoptosis of CD4?T cells in peripheral
blood was only measured at selected time points: at the initi-
ation of ART, at the end of treatment, and 28 days post-
treatment discontinuation. As shown in Fig. 4e, there was no
significant change in the percentage of apoptotic cells during
the follow-up, which confirmed our previous data showing no
discernible change in the apoptosis of CD4?T cells during
SIVagm infection of AGMs (48).
Dynamics of CCR5?CD4?T cells (target cells) during
ART. Our previous results showed that natural hosts of SIV
have significantly lower expression levels of CCR5 on their
CD4?T cells (47). Since CCR5?CD4?T cells are rapidly
depleted during HIV or SIV infection, it has been suggested
that the low levels of target cells might be a reason for the
nonpathogenicity of infection in natural hosts of SIV (62).
However, we recently showed that there is massive mucosal
CD4?T-cell depletion in acute SIV infection of natural hosts
(19, 48). Therefore, we analyzed the evolution of the CCR5?
CD4?T cells during ART. No significant change in the pop-
ulation of CCR5?CD4?T cells was observed during the first
10 days of therapy. However, a significant increase of CCR5?
CD4?T cells with an average slope of 0.11 cell/?l/day (P ?
0.0012) (Fig. 5) was observed between days 10 and 43 post-
In this study, we performed experimental SIVagm infections
of AGMs, treated the animals with potent antiretroviral treat-
ment during the chronic, steady-state SIVagm infection, and
measured the turnover of infected cells by applying the same
mathematical modeling previously used for pathogenic infec-
tions (52, 55, 79). We showed that the bulk of SIVagm repli-
cation during acute and chronic infection of AGMs occurs in
short-lived infected cells. Therefore, our results demonstrate
that the lack of disease progression in SIVagm infection in its
natural host is unlikely due to a slower virus and cell turnover
compared to pathogenic infections.
SIV infection in African NHPs that are natural hosts of SIVs
is characterized by the following: (i) high levels of viral repli-
cation during acute and chronic SIV infection in the same
range, if not higher, than in pathogenic HIV-1 infection of
humans and SIV infection of RMs (1, 43, 44, 46, 49–51, 65, 66);
(ii) good preservation of peripheral CD4?T cells for long
periods of time (46, 50, 66, 72); (iii) low levels of target cells
(CCR5?CD4?T cells) (47). Altogether, these characteristics
of SIV infection in natural hosts generated the hypothesis that
the lack of disease progression might be due to a more limited
impact of SIV replication on the homeostasis of CD4?T cells
(64). This limited impact could result from either (i) a longer
life span of the bulk of infected cells (activated effector mem-
ory CD4?T cells) or (ii) virus replication occurring in long-
lived cells (most likely macrophages). However, recent data
showed that the impact of SIV infection on CD4?T cells from
natural African NHP hosts during acute infection is very sim-
ilar to that observed during pathogenic infections (19, 48), with
FIG. 5. Dynamics of CCR5?CD4?T cells under antiretroviral treatment of AGMs chronically infected with SIVagm. A significant increase
was observed between day 10 of treatment and day 43 posttreatment.
3720PANDREA ET AL.J. VIROL.
early and severe depletion of mucosal CD4?T cells in both
AGMs and SMs (19, 48). This observation suggested that both
CD4?T-cell turnover and SIV cytopathicity are similar be-
tween progressive and nonprogressive infections.
With regard to the hypothesis that virus replication mainly
occurs in macrophages, there is some evidence against this: (i)
the dynamics of virus replication during acute infection is strik-
ingly similar between progressive and nonprogressive SIV in-
fections (51), which likely would not be the case if replication
were supported by long-lived infected cells that produce virus
slowly; (ii) the massive acute depletion of mucosal CD4?T
cells suggests that these cells are the SIV targets (19, 48);
moreover, although this massive mucosal CD4?T-cell deple-
tion involves all the CD4?T-cell subsets, the effector memory
CD4?T-cell pool is the most susceptible and shows only minor
restoration during chronic infection (19, 48), similar to patho-
genic HIV-1 and SIV infections (38, 56); (iii) we and others
(15) have provided direct evidence by in situ hybridization that,
during acute infection of AGMs, SIVagm replicates in lym-
phocytes and not in macrophages (Fig. 2) (47). Collectively,
these experimental results suggest that the in vivo biology of
SIV is comparable between progressive and nonprogressive
infections and that the bulk of virus replication also occurs in
short-lived infected cells during SIV infections of African NHP
hosts. However, the data presented thus far pertain only to
acute infection (when the virus can be studied by in situ tech-
niques). Moreover, the significant mucosal CD4?T-cell resto-
ration during chronic infection in the presence of high viral
replication indeed raises the question of whether or not the
bulk of viral replication during the chronic phase of infection
occurs in macrophages.
To address this question, we performed the current set of
experiments, to investigate the source of the virus during
chronic SIVagm infection of AGMs. The advantage of using
AGMs for these types of studies lies in the fact that, unlike
other African NHP species that are currently being used as
animal models for natural SIV infection (such as SMs and
mandrills), AGMs are not highly endangered and, therefore,
more complete studies can be carried out in this species. The
frequent testing during acute infection confirmed that the dy-
namics of SIVagm replication in experimentally infected
AGMs is very similar to those of other progressive and non-
progressive SIV infections (46, 48, 49, 51) and that during the
chronic infection there are negligible variations in the steady
state in SIVagm-infected AGMs.
In this study, chronic SIVagm-infected AGMs were admin-
istered a combination of two NRTIs: PMPA and FTC. The
choice of drugs was based on previous reports showing excel-
lent antiretroviral activity of these two compounds in monkeys
(28, 74). Moreover, other ARV classes, such as nonnucleoside
RT inhibitors, are not suitable for studies like these because
SIVs are intrinsically resistant to these drugs (78). Similarly,
SIVs reportedly have variable susceptibilities to protease in-
hibitors (14). Conversely, NRTIs are drugs of choice for these
types of studies because they are potent enough to induce
significant and rapid reduction in viral replication (28, 74) and
also because they do not affect preformed virus or the ability of
previously infected cells to continue to produce new virions
(11), thus allowing a reliable quantification of the in vivo turn-
over of infected cells and providing indirect information on
what cell type(s) supports virus replication in this nonpatho-
genic model of infection.
Administration of PMPA and FTC resulted in a rapid de-
cline of plasma VLs, which became undetectable in all six
animals. Interestingly, an initial rapid decline in plasma VL
observed during the first 18 h of treatment was followed by a
short viral rebound during the second day of treatment (Fig.
1c), probably the result of reduced drug exposure prior to the
establishment of steady-state pharmacokinetics (20). With con-
tinuous dosing, this rebound was followed by a sharp decline in
VLs, which became undetectable by the fourth day of treat-
ment. Our mathematical modeling of these data clearly indi-
cated that, in AGMs, the bulk (? ?90%) of SIVagm replication
occurred in cells with an average in vivo life span of 4 to 9.5 h,
shorter than estimated in HIV-1-infected humans and SIVmac-
infected RMs, when similar methods were used for the calcu-
lation (41, 55). This result suggests that SIVagm is cytopathic
in vivo for AGM-infected cells, as are HIV-1 for human cells
and SIVmac for RM cells. Similar data were reported for
another natural host of SIV, the sooty mangabey (18).
Previous studies carried out in HIV-infected humans and
SIV-infected macaques using different approaches reported
that lentiviral infection is characterized by rapid viral and in-
fected cell turnover. Initial studies, using HAART and mod-
eling the dynamics of viral replication control, estimated the
average half-life of virions at approximately 6 h and that for
infected cells at 1 day or less (55), and also the fraction of virus
replication occurring in short-lived cells (90 to 99%) versus
long-lived cells (1 to 10%) (53). More refined analyses,
through apheresis, have estimated an even faster viral clear-
ance in humans, with a viral half-life of approximately 20 to 45
min (58), and with more potent therapy, the half-life of virus-
producing cells was estimated at 0.7 days (37). In RMs, mea-
surements of viral clearance following bolus injection or con-
tinuous infusion showed that clearance was even faster in this
species, with a half-life of around 3.3 min (79). We note that in
the present study, the observed decay was so fast that we could
not reliably estimate the half-life of free virions in SIVagm-
We also estimated the basic reproduction number (R0) of
SIVagm in AGMs at 6.3 ? 1.3, considering that the intracel-
lular delay is ?1 day. This is similar to estimates for HIV in
humans (a harmonic mean of 13.9 in one study with n of 4 
and a mean of 5.4 in a study with n of 10 ), but probably
smaller than SIVsmE660 in pigtailed macaques (harmonic
mean, 28.9 ). This smaller R0is likely due to the faster loss
of infected cells in the AGM model, which is, for example, ?6
times faster than in the study on pigtailed macaque infection
referred to above.
At the cessation of antiretroviral therapy, the rebound in
viral replication was slower than during the acute infection,
occurring at 1 to 2 weeks post-treatment interruption to a set
point level that was strikingly similar to that observed prior to
therapy. This delay in virus rebound compared to the expo-
nential increase observed during acute infection may be due to
either the action of immune responses (which are absent in
primary infection) or to the lower availability of target cells
compared to primary infection. Interestingly, in sooty manga-
beys this rebound is faster (18).
ART induced only minor, although statistically significant,
VOL. 82, 2008 SIVagm DYNAMICS IN AFRICAN GREEN MONKEYS3721
changes in the number of peripheral CD4?T cells in AGMs
chronically infected with SIVagm. No significant changes in
CD4?T cells in the LNs and intestine were observed. This
difference with data reported in HIV-1-infected patients re-
ceiving HAART, in which there is an increase in the pool of
CD4?T cells (61), may be due to several factors: (i) AGMs
received only short-term ARV treatment, which might have
prevented us from observing a significant CD4?T-cell resto-
ration under treatment; (ii) in chronically infected AGMs, the
levels of peripheral CD4?T-cell counts are close to normal
levels, suggesting that chronic virus replication does not induce
peripheral CD4?T-cell depletion; our previous data showing
that mucosal CD4?T cells can be restored in AGMs chroni-
cally infected with SIVagm (48) support these results; (iii) it
has been reported that in humans, the initial CD4?T-cell
rebound during HAART is also due to redistribution of CD4?
T cells from lymphoid tissues (6, 8); it is possible that in natural
hosts, the CD4?T-cell redistribution under ART is less sig-
nificant, as a consequence of less impact of infection on im-
mune parameters, such as T-cell activation and proliferation;
(iv) finally, in HIV-1-infected humans, CD4?T-cell restora-
tion is associated with a significant decrease in the levels of
immune activation (8). In SIV natural hosts, immune activa-
tion, proliferation, and apoptosis markers are normal during
chronic infection (7, 30, 44, 46, 48, 50, 64–66, 73) and, as we
have shown here, not significantly influenced by ART, which
may also explain the lack of significant immunologic changes in
the homeostasis of CD4?T cells in ARV-treated AGMs. Note
that only minor increases in CD4?T-cell proliferation were
observed in our AGMs and that these were correlated with the
rebound in viral replication after the cessation of therapy.
In conclusion, our results show that, in chronically infected
AGMs, SIVagm replication occurs in short-lived infected cells,
thus extending our previous observation of SIVagm replication
in lymphocytes and not in macrophages during acute infection
(48). Our results corroborate those obtained in another animal
model for natural SIV infection, the sooty mangabey (18), and
together suggest the bulk of virus replication occurs in acti-
vated CD4?T cells in African natural hosts of SIV. Therefore,
the lack of disease progression in natural hosts is likely not due
to a prolonged survival of infected cells and/or a reduced
intrinsic cytopathicity of SIVs. Since the in vivo dynamics of
chronic lentiviral infection is very similar between progressive
and nonprogressive infections, the outcome of infection must
be dependent on other factors, such as immune activation
and/or other unknown factors.
We thank Andrew Lackner and Preston Marx for helpful discussions
and the veterinary staff of the Tulane National Primate Research
Center for their assistance with the animal studies. PMPA and FTC
were kindly provided by Gilead Sciences.
This work was supported by NIH grants RO1 AI064066 and
R21AI069935 (I.P.), RO1 AI065325 (C.A.), RR06555 and AI28433
(A.S.P.), RR18745 (R.M.R.), and RR-00168 (Tulane National Primate
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