Simian immunodeficiency virus SIVagm dynamics in African green monkeys.
ABSTRACT 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 (tenofovir) 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.
- SourceAvailable from: David C Montefiori[Show abstract] [Hide abstract]
ABSTRACT: The design of an effective vaccine to reduce the incidence of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) via breastfeeding will require identification of protective immune responses that block postnatal virus acquisition. Natural hosts of simian immunodeficiency virus (SIV) sustain nonpathogenic infection and rarely transmit the virus to their infants, despite high milk virus RNA loads. This is in contrast to HIV-infected women and SIV-infected rhesus macaques (RhMs), non-natural hosts which exhibit higher rates of postnatal virus transmission. In this study, we compared the systemic and mucosal B cell responses of lactating, SIV-infected African green monkeys (AGMs), a natural host species, to that of SIV-infected RhMs and HIV-infected women. AGMs did not demonstrate hypergammaglobulinemia or accumulate circulating memory B cells during chronic SIV infection. Moreover, the milk of SIV-infected AGMs contained higher proportions of naive B cells compared to RhMs. Interestingly, AGMs exhibited robust milk and plasma Env binding antibody responses that were one to two logs higher than in RhMs and humans and demonstrated autologous neutralizing responses in milk at one year post infection. Furthermore, the plasma and milk Env gp120-binding antibody responses were equivalent to or predominant over Env gp140-binding antibody responses in AGMs, in contrast to that in RhMs and humans. The strong gp120-specific, functional antibody responses in the milk of SIV-infected AGMs may contribute to the rarity of postnatal transmission observed in natural SIV hosts.Journal of Virology 08/2013; · 5.08 Impact Factor
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ABSTRACT: The hallmark of acquired immunodeficiency syndrome (AIDS) pathogenesis is a progressive depletion of CD4(+) T-cell populations in close association with progressive impairment of cellular immunity and increasing susceptibility to opportunistic infections (OI). Disease progression in untreated human immunodeficiency virus (HIV) infection can take many years, and it was originally hypothesized to be a consequence of slow, viral-mediated CD4(+) T-cell destruction. However, massive CD4(+) memory T-cell destruction is now known to occur quite early in infection, almost always without overt immunodeficiency. In most individuals, this initial destruction is countered by CD4(+) memory T-cell regeneration that preserves CD4(+) T-cell numbers and functions above the threshold associated with overt immunodeficiency. This regeneration, which occurs in the setting of chronic immune activation and immune dysregulation does not, however, restore all functionally important CD4(+) T-cell populations and is not stable over the long term. Ultimately, CD4(+) memory T-cell homeostasis fails and critical effector populations decline below the level necessary to prevent OI. Thus, the onset of overt immune deficiency appears to be intimately linked with CD4(+) memory T-cell dynamics and reflects the complex interplay of direct viral cytopathogenicity and the indirect effects of persistent immune activation on CD4(+) memory T-cell proliferation, differentiation, and survival.Immunological Reviews 07/2013; 254(1):54-64. · 12.16 Impact Factor
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ABSTRACT: We assessed the role of myeloid dendritic cells (mDCs) in the outcome of SIV infection by comparing and contrasting their frequency, mobilization, phenotype, cytokine production and apoptosis in pathogenic (pigtailed macaques, PTMs), nonpathogenic (African green monkeys, AGMs) and controlled (rhesus macaques, RMs) SIVagmSab infection. Through the identification of recently replicating cells, we demonstrated that mDC mobilization from the bone marrow occurred in all species postinfection, being most prominent in RMs. Circulating mDCs were depleted with disease progression in PTMs, recovered to baseline values after the viral peak in AGMs, and significantly increased at the time of virus control in RMs. Rapid disease progression in PTMs was associated with low baseline levels and incomplete recovery of circulating mDCs during chronic infection. mDC recruitment to the intestine occurred in all pathogenic scenarios, but loss of mucosal mDCs was associated only with progressive infection. Sustained mDC immune activation occurred throughout infection in PTMs and was associated with increased bystander apoptosis in blood and intestine. Conversely, mDC activation occurred only during acute infection in nonprogressive and controlled infections. Postinfection, circulating mDCs rapidly became unresponsive to TLR7/8 stimulation in all species. Yet, stimulation with LPS, a bacterial product translocated in circulation only in SIV-infected PTMs, induced mDC hyperactivation, apoptosis and excessive production of proinflammatory cytokines. After infection, spontaneous production of proinflammatory cytokines by mucosal mDCs increased only in progressor PTMs. We thus propose that mDCs promote tolerance to SIV in the biological systems that lack intestinal dysfunction. In progressive infections, mDC loss and excessive activation of residual mDCs by SIV and additional stimuli, such as translocated microbial products, enhance generalized immune activation and inflammation. Our results thus provide a mechanistic basis for the role of mDCs in the pathogenesis of AIDS and elucidate the causes of mDC loss during progressive HIV/SIV infections.PLoS Pathogens 10/2013; 9(10):e1003600. · 8.14 Impact Factor
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 consisted ofplasma
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).
obtainedfrom an experimentally
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
3714 PANDREA 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, 2008 SIVagm 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