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IMMUNOBIOLOGY
Suppression of adaptive immune responses during primary SIV infection of
sabaeus African green monkeys delays partial containment of viremia but does not
induce disease
Roland C. Zahn,1 Melisa D. Rett,1 Ming Li,2 Haili Tang,2 Birgit Korioth-Schmitz,1 Harikrishnan Balachandran,1 Robert White,3
Sarah Pryputniewicz,4 Norman L. Letvin,1 Amitinder Kaur,4 David C. Montefiori,2 Angela Carville,5 Vanessa M. Hirsch,6
Jonathan S. Allan,3 and Jörn E. Schmitz1
1Division
of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Laboratory for AIDS Vaccine Research and
Development, Department of Surgery, Duke University Medical Center, Durham, NC; 3Department of Virology and Immunology, Southwest Foundation for
Biomedical Research, San Antonio, TX; 4Division of Immunology and 5Division of Primate Resources, New England Primate Research Center, Harvard Medical
School, Southborough, MA; and 6Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD
One of the most puzzling observations in
HIV research is the lack of pathogenicity
in most nonhuman primate species that
are natural hosts of simian immunodeficiency virus (SIV) infection. Despite this,
natural hosts experience a level of viremia similar to humans infected with HIV
or macaques infected with SIV. To determine the role of adaptive immune responses in viral containment and lack of
disease, we delayed the generation of
cellular and humoral immune responses
by administering anti-CD8– and antiCD20 lymphocyte–depleting antibodies to
sabaeus African green monkeys (Chlorocebus sabaeus) before challenge with
SIVsab9315BR. In vivo lymphocyte depletion
during primary infection resulted in a
brief elevation of viremia but not in disease. Based on the magnitude and timing
of SIV-specific CD8ⴙ T-cell responses in
the lymphocyte-depleted animals, CD8ⴙ
T-cell responses appear to contribute to
viral containment in natural hosts. We
found no evidence for a contribution of
humoral immune responses in viral containment. These studies indicate that natural hosts have developed mechanisms in
addition to classic adaptive immune responses to cope with this lentiviral infection. Thus, adaptive immune responses
in natural hosts appear to be less critical
for viral containment than in HIV infection. (Blood. 2010;115(15):3070-3078)
Introduction
AIDS virus infections in most non-natural hosts, including humans
and Asian nonhuman primates, ultimately result in overt signs of
disease and immune failure. However, there is strong evidence that
adaptive immune responses contribute to partial containment of
viremia in these species.1 Correlative investigations have shown
that the early containment of viremia in humans and rhesus
macaques coincides with the expansion of AIDS virus-specific
CD8⫹ T-cell responses.2-4 More direct evidence for the role of
CD8⫹ T cells in viral containment was obtained through in vivo
depletion experiments in nonhuman primates, such as simian
immunodeficiency virus (SIV) infection of rhesus macaques.5,6
Furthermore, SIV-challenged rhesus macaques vaccinated with
vaccines that primarily elicit CD8⫹ T-cell responses experience
more efficient viral containment and significantly longer survival
than sham-vaccinated control animals.7
The role of humoral immune responses is more difficult to
evaluate. HIV-specific B cells produce HIV envelope-specific
antibodies as early as 2 weeks after infection.8 However, neutralizing antibodies (NAbs) develop only after months and are initially
highly specific for the autologous transmitted/founder virus. NAbs
also mostly fail to neutralize the contemporaneous virus during
chronic infection because the virus rapidly evolves to escape
autologous neutralization.9,10 Similarly, rhesus macaques infected
with SIV are slow to develop NAbs and experience continued viral
escape.11,12 However, the potential potency of humoral immune
responses has been demonstrated by complete protection of rhesus
macaques from pathogenic SHIV89.6P challenge after passive
transfer of broadly NAbs.13,14 Recently, a limited number of studies
have evaluated the role of B cell–mediated immune responses in
SIV containment in rhesus macaques.15-17 Humoral immune responses did not contribute to viral containment in primary infection,15 but an accelerated disease progression was observed in some
studies when B-cell function was impaired by anti-CD20 antibody
administrations, suggesting that B cells are important during the
chronic stages of infection.16
At least 40 different African apes and monkey species are
natural hosts of lentivirus infections. Unlike humans and
macaques, these animals only rarely show signs of disease.18,19
The general lack of pathogenicity in these species is still not
completely understood. Recent studies in natural hosts of SIV
demonstrate a similar dynamic of primary viremia, level of peak
viremia, and eventual set point viremia compared with pathogenic models.20,21 Natural hosts, such as African green monkeys
(AGMs) and sooty mangabeys, appear to have undergone
evolutionary adaptations that enable these species to better cope
with AIDS virus infections. These adaptations include the lack
Submitted October 8, 2009; accepted January 22, 2010. Prepublished online
as Blood First Edition paper, February 10, 2010; DOI 10.1182/blood-2009-10245225.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
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BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
of chronic immune activation,22,23 a paucity of SIV target cells
(CCR5⫹CD4⫹ T cells)24 and the apparent presence of a unique
subset of T cells that provide helper function in the absence of
CD4 expression.25,26
Prior studies suggest that adaptive immune responses seem to
play a limited role in containing SIV in chronic infection in natural
hosts.27,28 CD8⫹ T-cell responses are generally less robust than in
pathogenic SIV infection and do not correlate with plasma viral
load.28-30 Furthermore, CD8⫹ lymphocyte depletion in chronic
infection in AGMs and sooty mangabeys resulted in a variable
outcome with modest increase in viremia in some studies31 (R.C.Z.,
M.D.R., E. Hagan, R.W., A.C., V.M.H., M. C. Gauduin, J.S.A., and
J.E.S., unpublished observations, September 2009) and a more
significant increase in another study (A.K., Z. Wang, N. Kassis,
R. M. Ribeiro, S.P., R. P. Johnson, K. Reimann, J.E.S., A. S.
Perelson, H. M. McClure, and S. P. O’Neill, unpublished observations, October 2003). Humoral immune responses in chronic
infection are mainly directed against Env, and only low titers of
NAbs are elicited.27,32
Here, we used sabaeus AGMs (Chlorocebus sabaeus) to
evaluate the role of the early development of adaptive immune
responses in viral containment and lack of disease progression. We
depleted CD8⫹ lymphocytes and B cells in vivo with monoclonal
antibodies (mAb) to delay the generation of both CD8⫹ T-cell and
humoral immune responses during primary infection.
Methods
Animals and viruses
A total of 12 sabaeus AGMs (C sabaeus) were recruited for this study. Three
animals were imported from St Kitts in the Caribbean and 9 were purchased
from New Iberia, LA. The animals were infected with an equivalent of
143 ng of p27 tissue-culture supernatant of Molt4(cl8) cells infected with
SIVsab9315BR. This virus was originally isolated from cell-free homogenized
brain fluid from a naturally infected sabaeus AGM.33 Lymph nodes were
obtained by excisional biopsy under anesthesia with Telazol. For all other
procedures, animals were sedated with ketamine hydrochloride. All animals
were maintained in accordance with the guidelines of the Committee on the
Care and Use of Laboratory Animals under a National Institute of Allergy
and Infectious Diseases (NIAID)–approved animal study protocol,34 and all
studies and procedures were reviewed and approved by the Institutional
Animal Care and Use Committees of the National Institutes of Health
(NIH) and Harvard University.
CD8ⴙ and CD20ⴙ lymphocyte depletion
Six AGMs were transiently depleted of CD8⫹ and CD20⫹ lymphocytes by
the administration of the chimeric anti–human CD8␣ mAb, cM-T807 (NIH
Nonhuman Primate Reagent Resource) and the anti–human CD20 mAb,
Rituxan (rituximab), purchased from Genentech. cM-T807 was administered at 10 mg/kg body weight subcutaneously on day 0 (the day of SIV
infection) followed by 5 mg/kg intravenous injections on days 3 and
7. Rituximab was administered intravenously at 50 mg/kg body weight on
days ⫺7, 14, and 35. One dose of human immunoglobulin (IgIV; NIH
Nonhuman Primate Reagent Resource) was administered intravenously at
50 mg/kg on days 0 to 6 AGMs to serve as a control.
The effect of the lymphocyte-depleting antibody treatment and SIV
infection on lymphocyte subset changes was determined by flow cytometric
investigations using an LSR II instrument (BD Biosciences) and FlowJo
software (TreeStar; supplemental Methods, available on the Blood website;
see the Supplemental Materials link at the top of the online article).
ROLE OF SIV-SPECIFIC IMMUNE RESPONSES IN AGM
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Plasma viral load assay
In brief, SIVsab9315BR plasma RNA levels were quantified by the
Ultrasense One-Step Quantitative RT-PCR System (Invitrogen; also see
supplemental Methods).
Amplification and cloning of the envelope and rev SIVsab9315BR
DNA cassettes and serum-NAb assays
In brief, Env-rev cassettes were cloned from DNA obtained from cultured
peripheral blood mononuclear cells (PBMCs) after infection with an animal
challenge stock of molecularly cloned SIVsab9315BR. An assay stock of
molecularly cloned SIVsab92315BR Env-pseudotyped viruses was prepared by
transfection in 293T cells and was titrated in TZM-bl cells as described.35
Neutralization was measured as a function of reduction in luciferase
reporter gene expression after a single round of infection in TZM-bl cells as
described35 (also see supplemental Methods).
Cellular immune response
The interferon-␥ (IFN-␥) ELISpot assay and the intracellular cytokine
staining (ICS) assay was performed as previously described.29 The ICS was
modified to accommodate a PBMC stimulation time of 9 hours that permits
a greater sensitivity to detect cytokine responses compared with a 6-hour
incubation period. The peptide pools for stimulation of AGM-derived
PBMC in both assays consisted of overlapping 15-mer peptides spanning
the SIVsab92018 Env protein or the Gag protein. A total of 2 ␮g/mL
SIVsab92018 Gag pool or 2 ␮g/mL SIVsab92018 Env peptide pools (Mimotopes,
and NIH/NIAID Reagent Resource Support Program for AIDS Vaccine
Development, Quality Biological; R. L. Brown, principal investigator) was
used for PBMC stimulations.
Statistical analyses
Statistical analyses and graphical presentations were computed with
GraphPad Prism, Version 5.02 (GraphPad Prism software). P values of less
than .05 were considered significant. Mann-Whitney tests were applied for
comparison of 2 groups. A Spearman correlation test was performed to analyze
the association between plasma viral RNA loads and various parameters
(including absolute CD4⫹ T-cell counts and memory CD4⫹ T cells).
Results
Administration of cM-T807 and rituximab to sabaeus AGMs
induces temporal depletion of CD8ⴙ and CD20ⴙ lymphocytes in
peripheral blood and lymphatic tissues
To assess the role of adaptive immune responses in the control of
SIV infection in sabaeus AGMs, we used CD8⫹ and CD20⫹
lymphocyte depletion to temporarily delay adaptive immune
responses during primary SIVsab9315BR infection in 6 AGMs. A
control group of 6 animals was also inoculated with SIVsab9315BR
but received IgIV instead of the lymphocyte-depleting antibodies.
The CD8⫹ lymphocyte depletion resulted in an efficient elimination of CD8⫹ T cells in peripheral blood for 3 weeks in 5 of
6 animals (Figure 1B). A brief depletion of CD8⫹ T cells (1 week)
was observed in 1 animal (no. 364). Similarly, we observed a
near-total depletion of CD8⫹ T cells in lymph nodes at day 14 after
infection (Figure 1D). As CD8⫹ T cells reappeared in peripheral
blood, CD8⫹ T cells also reappeared in lymph nodes on weeks 5
and 10 after infection (Figure 1D). In contrast, significant changes
in CD8⫹ T cells were not observed in the 6 IgIV-treated control
AGMs (Figure 1A,C). Interestingly, all of the AGMs with efficient
CD8⫹ lymphocyte depletion had a transient 2.5- to 5.0-fold
(median, 4.2-fold) increase of CD8⫹ T-cell counts for 3 to 13 weeks
after the reappearance of CD8⫹ T cells. The relatively high levels
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BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
Figure 1. CD8ⴙ T-cell and NK-cell depletion in SIV-infected AGMs.
Absolute CD8⫹ T cells in peripheral blood (A-B) and lymph nodes
(C-D) and peripheral blood NK-cell (E-F) counts in 12 sabaeus African
green monkeys (AGMs) infected intravenously with SIVsab9315BR. Six
AGMs received 1 subcutaneous dose of 10 mg/kg of the anti-CD8␣
mAb cM-T807 on day 0 before simian immunodeficiency virus (SIV)
infection and 2 intravenous doses of 5 mg/kg cM-T807 on days 3 and
7 after infection to deplete CD8⫹ lymphocytes (B,D,F). These
6 animals also received 50 mg/kg anti-CD20 rituximab antibody
intravenously at days ⫺7, 14, and 35 after infection to deplete B cells.
The other 6 animals received 1 intravenous injection of 50 mg/kg IgIV
7 days before infection to serve as control animals (A,C,E). The 2 in
panels B, D, and F indicate the injection of the CD8⫹ lymphocytedepleting mAb. CD8⫹ T cells were gated as CD8⫹ CD3⫹ lymphocytes
and NK cells as CD8⫹ CD3⫺ lymphocytes. Absolute CD8⫹ T-cell and
NK-cell numbers were calculated from white blood cell counts. p.i.
indicates postinfection.
of CD8⫹ T cells slowly returned to pretreatment levels, with the
exception of animal no. 366, which maintained high levels of
CD8⫹ T cells until week 42 after infection. This massive expansion
of CD8⫹ T cells on reappearance has not been observed in CD8⫹
lymphocyte depletion experiments in either noninfected or acutely
SIV-infected rhesus monkeys.5,36,37
Similar to humans and rhesus monkeys, AGMs harbor 2 distinct
subsets of CD8⫹ T cells: CD8␣␣ homodimer and CD8␣␤ heterodimer expressing cells.29 The CD8⫹ T cells that first reappeared
after CD8⫹ lymphocyte depletion in the 5 well–depleted AGMs
were mainly CD8␣␣⫹ T cells (5.5- to 21.2-fold increase from
levels before depletion; median, 11.3-fold). CD8␣␤⫹ T cells
reappeared much slower and did not reconstitute to predepletion
levels in 5 of 6 CD8⫹ and CD20⫹ lymphocyte-depleted AGMs
during the observation period of 42 weeks after SIV infection (data
not shown).
Figure 2. B-cell depletion in SIV-infected AGMs. Six sabaeus
AGMs were depleted by mAb cM-T807 of CD8⫹ lymphocytes and by
mAb rituximab of CD20⫹ lymphocytes (B,D), and 6 AGMs received
purified control Ab IgIV (A,C). Absolute B-cell number in peripheral
blood (A-B) and percentage of B cells in lymph node cell suspensions
(C-D). The 2 in panels B and D indicate the injection time points of the
CD20⫹ lymphocyte-depleting mAb rituximab. p.i. indicates postinfection.
Similar to rhesus monkeys,38 natural killer (NK) cells from
AGMs show a uniformly high expression of the CD8 molecule
(data not shown). Therefore, as expected, cM-T807 administrations
also led to a depletion of NK cells. In general, the duration of
NK-cell depletion was comparable with the CD8⫹ T-cell depletion
(Figure 1F). In contrast, the 6 control AGMs did not show a
significant change in the number of NK cells after SIV infection
and control Ab administration (Figure 1E).
B-cell depletion in peripheral blood was efficient in 4 of
6 CD8⫹ and CD20⫹ lymphocyte-depleted AGMs and lasted for 8 to
14 weeks after infection (Figure 2B). Two AGMs (no. 361 and no.
364) responded inefficiently to rituximab administration and experienced a short-term B-cell depletion of only 1 week. In contrast to
CD8⫹ lymphocyte counts, none of the animals showed an increase
in B-cell counts above pre-CD20⫹ lymphocyte depletion values
after reappearance of B cells in peripheral blood. The control
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BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
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animals experienced a transient decrease in B-cell counts coincident with the peak of viremia, 2 weeks after infection (Figure 2A).
The pattern of B-cell depletion in lymph nodes (Figure 2D) was
similar to what was observed in peripheral blood. The 3 animals
with the longest B-cell depletion (no. 365, no. 366, and no. 368)
had a continuous decline of B cells, and the percentage of B cells in
lymphocytes obtained from these lymph nodes dropped to 3% at
10 weeks after infection. The other 3 CD8⫹ and CD20⫹ lymphocytedepleted AGMs experienced only a transient B-cell depletion at
week 2 after infection but did not show a long-lasting depletion in
the lymph node. In contrast, the relative percentage of B cells in
lymph nodes increased after SIV infection in control animals
(median before SIV infection, 23.3%; range, 19.4%-36.6%; median
week 10 after infection, 39.7%; range, 35.89%-45.7%; Figure 2C).
CD8ⴙ and CD20ⴙ lymphocyte depletion in SIV-infected AGMs
results in a brief temporal delay of partial viral containment
The kinetics of plasma viral RNA levels in the control animals was
comparable with that of pathogenic SIVmac infection of rhesus
monkeys, although the magnitude of the viremia in AGM was
generally lower. All control Ab-treated AGM experienced a peak of
viremia at 2 weeks after infection followed by a nadir. Set-point
viremia was established by 5 to 6 weeks after infection with a
median of 2.5 ⫻ 104 SIV RNA copies per milliliter (range, 2 ⫻ 102
to 7.4 ⫻ 104 SIV RNA copies/mL; Figure 3A). Plasma viral RNA
levels of the CD8⫹ and CD20⫹ lymphocyte-depleted AGMs were
similar to the control AGMs at 2 weeks after infection and at
set-point (median, 2.8 ⫻ 104 SIV RNA copies/mL; range, 3 ⫻ 102
to 9.3 ⫻ 104 SIV RNA copies/mL; Figure 3B). However, SIV RNA
copies remained high until 3 weeks after infection and subsequently decreased until week 6 after infection. Thus, SIV RNA in
plasma was significantly higher in CD8⫹ and CD20⫹ lymphocytedepleted AGMs at weeks 3 and 4 compared with the control AGMs
(Figure 3C). Despite this prolonged peak of viremia, the levels of
set-point viremia did not differ between the 2 groups. AGM no.
364, which showed inefficient depletion, did not experience
prolonged peak viremia and was more comparable with the control
sabaeus AGMs (Figure 3B).
SIV-specific cellular immune responses are significantly
increased in CD8ⴙ and CD20ⴙ lymphocyte-depleted AGMs after
reappearance of CD8ⴙ lymphocytes
The postpeak viremia decline of viral RNA in CD8⫹ and CD20⫹
lymphocyte-depleted AGMs coincided with the reappearance of
CD8⫹ lymphocytes, suggesting that CD8⫹ lymphocytes partially
contain virus replication in AGMs. Therefore, we examined the
magnitude of SIV-specific adaptive immune responses in both
groups of AGMs.
First, we investigated IFN-␥ production of total PBMCs after
stimulation with SIVsab Gag and Env 15-mer peptide pools in an
ELISpot assay. Control Ab-treated AGMs had a range of Gagspecific IFN-␥ responses between 0 and 720 spot-forming cells
(SFC)/106 PBMCs (median, 228 SFC/106 PBMCs; Figure 4A) and
Env-specific IFN-␥ responses between 0 and 790 SFC/106 PBMCs
(median, 315 SFC/106 PBMCs; Figure 4C). Gag and Env responses
were first detectable in the control group at week 4 and were
sustained throughout the study. CD8⫹ and CD20⫹ lymphocytedepleted AGMs, however, had significantly higher IFN-␥ responses at 4 weeks after infection after stimulation with SIVsab Gag
(range, 940-1290 SFC/106 PBMCs; median, 1020 SFC/106 PBMCs; P ⫽ .005, Mann-Whitney test; Figure 4B) and SIVsab Env
Figure 3. Temporally increased plasma SIV viremia in CD8ⴙ and CD20ⴙ
lymphocyte-depleted SIV-infected AGMs. SIVsab9315BR RNA copy numbers were
determined by quantitative PCR in control Ab-treated AGMs (A) and CD8⫹ and
CD20⫹ lymphocyte-depleted AGMs (B). Median viral RNA copy number for both
groups (C). *Statistically significant difference between the control (solid line, F) and
CD8⫹ and CD20⫹ lymphocyte-depleted group (dashed line, 䡺) at the indicated time
points (Mann-Whitney test, P ⱕ .05). p.i. indicates postinfection.
peptide pools (range, 390-5705 SFC/106 PBMCs; median, 3385
SFC/106 PBMCs; P ⫽ .009, Mann-Whitney test; Figure 4D). The
high magnitude of IFN-␥ production by CD8⫹ and CD20⫹
lymphocyte-depleted AGMs eventually declined and reached levels comparable with those observed in the control group. Interestingly, the inefficiently depleted AGM no. 364 did have a similar
magnitude of IFN-␥ response after Env peptide stimulation as the
control AGMs.
To further assess the cytokines produced after CD8⫹ T cells
reappeared, we performed ICS after stimulation with SIVsab Gag
and Env peptide pools. We found only very low responses
(⬍ 0.15% of CD4⫹ T cells) in the CD4⫹ T-cell subset without a
significant difference between the groups of AGMs (data not
shown). In CD8⫹ T cells, the strongest SIV-specific responses were
observed by IFN-␥ production (Figure 5A,D,G) followed by tumor
necrose factor (TNF-␣) production (Figure 5C,F,I). Relatively
weak SIV-specific CD8⫹ T-cell responses were seen by interleukin-2
(IL-2) production (Figure 5B,E,H). The CD8⫹ T-cell responses
showed a pattern of IFN-␥ expression after peptide stimulation
similar to the ELISpot assay, albeit at a lower sensitivity. Gag as
well as Env peptide pool stimulation led to a 20- to 150-fold higher
median number of IFN-␥–expressing CD8⫹ T cells/␮L in the
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BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
Figure 4. Increased IFN-␥ immune responses in CD8ⴙ and CD20ⴙ
lymphocyte-depleted SIV-infected AGMs. SIVsab IFN-␥ cytokine
release of PBMCs obtained from 12 SIV-infected sabaeus AGMs.
PBMCs from 6 control Ab-treated AGMs (A,C) and 6 CD8⫹ and
CD20⫹ lymphocyte-depleted AGMs (B,D) were stimulated with SIVsab
Gag and Env 15-mer peptide pools for 18 hours at 37°C. The values
given represent the number of IFN-␥ SFCs in 106 PBMCs in response
to stimulation with the Gag (A-B) or Env peptide pool (C-D). Responses were considered positive if their value was greater than
2 times the SD of the mean of the media control wells. All values were
background corrected using the media controls. p.i. indicates postinfection.
CD8⫹ and CD20⫹ lymphocyte-depleted AGMs than in the control
AGMs (Figure 5G). Again, stimulation with Env (Figure 5D)
resulted in a higher number of IFN-␥–producing cells than
stimulation with Gag peptides (Figure 5A) in the CD8⫹ and CD20⫹
lymphocyte-depleted group (week 4, P ⬎ .082; week 5, P ⬍ .066;
week 10, P ⬎ .093, week 14, P ⬎ .128; Mann-Whitney test). The
number of IFN-␥⫹CD8⫹ T cells in the 2 groups of AGM was not
significantly different after 18 weeks after infection.
As seen with the IFN-␥ responses, TNF-␣ responses were
significantly higher in the CD8⫹ and CD20⫹ lymphocyte-depleted
animals than in the control group at weeks 4 and 5 after infection
(Gag week 4, P ⬍ .045; Gag week 5, P ⬍ .007; Env week 4,
P ⬍ .017; Env week 5, P ⬎ .004; Mann-Whitney test; Figure
5C,F,I). IL-2 responses also trended higher in CD8⫹ and CD20⫹
lymphocyte-depleted animals compared with the control animals
(Gag week 4, P ⬍ .158; Gag week 5, P ⬍ .091; Env week 4,
P ⬎ .104; Env week 5, P ⬎ .146; Mann-Whitney test; Figure
Figure 5. Increased SIV-specific expression of IFN-␥, IL-2, and
TNF-␣ cytokine responses in CD8ⴙ T cells from CD8ⴙ and CD20ⴙ
lymphocyte-depleted SIV-infected AGMs. Intracellular expression
of IFN-␥ (A,D,G), IL-2 (B,E,H), and TNF-␣ (C,F,I) in SIVsab Gag and
Env peptide pool-stimulated CD8⫹ T cells. Freshly isolated PBMCs
were stimulated for 9 hours at 37°C in the presence of either Env or
Gag peptide pools. The intracellular staining in CD8⫹ T cells stimulated with Gag or Env peptide pools is shown for control Ab-treated
AGMs in black and for CD8⫹ and CD20⫹ lymphocyte-depleted AGMs
in red (A-F). (G-I) Median responses of the 6 control AGMs in green
and of the 6 CD8⫹ and CD20⫹ lymphocyte-depleted AGMs in blue.
*Statistically significant difference between the control and CD8⫹ and
CD20⫹ lymphocyte-depleted group at the indicated time points (MannWhitney test, P ⱕ .05). Responses were considered positive if their
value was greater than 2 times the SD of the mean of the media control
wells. All values were background corrected using the media controls.
Responses and background were determined separately for the
different cytokines. p.i. indicates postinfection.
5B,E,H). As observed with the IFN-␥ responses, the magnitude of
Env peptide pool TNF-␣ and IL-2 responses was higher than the
Gag peptide pool responses (TNF-␣ week 4, P ⫽ .09; TNF-␣ week
5, P ⫽ .06; IL-2 week 4, P ⬍ .225; IL-2 week 5, P ⬎ .463;
Mann-Whitney test; Figure 5B,C,E-F,H-I). These results demonstrate that SIV-infected AGMs are capable of generating highmagnitude cellular immune responses. Furthermore, CD8⫹ T-cell
immune responses are very probably involved in partial containment of SIV viremia.
Administration of rituximab delays the generation of NAbs in
SIV-infected sabaeus AGMs
We and others have previously shown that AGMs and other natural
hosts exhibit relatively low levels of SIV-specific NAbs.19,27,39
Using an SIVsab9315BR Env-pseudotyped HIV-1 virus to measure
NAb activity, we were able to reproducibly detect SIVsab9315BR
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Figure 6. Delayed appearance of SIV-specific humoral immune responses has no effect on set-point viremia of SIV-infected AGMs. SIVsab9315BR NAb titers in control
Ab-treated AGMs (A) and CD8⫹ and CD20⫹ lymphocyte-depleted AGMs (B). The median NAb titer was calculated for both groups (C). *Statistically significant difference
between the control (solid line, F) and CD8⫹ and CD20⫹ lymphocyte-depleted group (dashed line, 䡺) at the indicated time points (Mann-Whitney test, P ⱕ .05). The 2 animals
with the most efficient/longest B-cell depletion, no. 365 (D) and no. 366 (E), in the group of antibody-treated and SIV-infected sabaeus AGMs are shown. The appearance of
NAbs (red lines) coincided with the reappearance of peripheral blood B cells (blue lines) but had no effect on set-point viremia. p.i. indicates postinfection.
NAbs in all AGMs investigated (Figure 6). NAbs were first
detected in control AGMs at weeks 3 to 7 and reached peak levels
of NAb titers of 1.5 to 4.7 ⫻ 104 (median, 3.7 ⫻ 104, Figure 6A).
In contrast, the CD8⫹ and CD20⫹ lymphocyte-depleted AGMs
developed SIV NAb responses significantly later at weeks 7 to 20
(P ⫽ .02; Mann-Whitney test) but eventually reached levels similar to those observed in the control animals (range, 0.4-9.2 ⫻ 104;
median, 3.8 ⫻ 104; Figure 6B). The 2 AGMs with the longest
B-cell depletion (AGMs no. 365 and no. 366) also exhibited the
most profound delay in SIV NAb responses. In weeks 5 to 22 after
infection (except week 15 after infection), the SIV NAb responses
in B cell–depleted AGMs were significantly lower compared with
the control animals (Figure 6C). Additional screening for SIV
antibodies by Western blot of AGMs serum against whole
SIVsab9315BR lysate confirmed the results from the NAb assay (data
not shown). Taken together, the data obtained by SIV-specific NAb
assay demonstrated a solid reduction in humoral immune responses
(NAb titer ⬍ 102) during the first 10 weeks in 4 AGMs (no. 364,
no. 365, no. 366, and no. 368). However, the appearance of NAbs
did not have a profound effect on the magnitude of viremia (Figure
3C). This is particularly evident in the 2 animals with the longest
B-cell depletion (animals no. 365 and no. 366), which showed a
relatively stable set-point viremia for more than 30 weeks, although
a significant difference in the level of NAb was observed during
this time period (Figure 6D-E).
CD8ⴙ and CD20ⴙ lymphocyte depletion does not affect the
number of memory and Ki-67ⴙ CD4ⴙ T cells or lead to the
induction of an AIDS-like disease in SIV-infected AGMs
One feature of nonpathogenic AIDS virus infection is the maintenance of relatively stable CD4⫹ T-cell counts, whereas pathogenic
AIDS virus infection is characterized by a progressive loss of
CD4⫹ T cells. We observed an initial small gain of memory CD4⫹
T cells until 1 week after infection (Figure 7A) in 5 of the 6 control
AGMs. After this transient increase, memory CD4⫹ T-cell numbers
decreased by 36% at peak viremia (2 weeks after infection). The
levels of memory CD4⫹ T cells did not recover to preinfection
values but remained stable over weeks 6 to 42 after infection
(median before infection, 140 cells/␮L; median after infection,
107 cells/␮L, P ⫽ .009, Mann-Whitney test). A similar phenomenon was observed in the lymphocyte-depleted AGMs (median
before infection, 151 cells/␮L, median after infection, 128 cells/␮L,
P ⫽ .013, Mann-Whitney test, Figure 7B). Memory CD4⫹ T-cell
numbers did not differ significantly between the 2 groups at any
time point (Figure 7C).
Similarly, no statistically significant difference was detected in
the fold change of the number of Ki-67⫹CD4⫹ T cells between both
groups of animals (except on 1 time point at week 5 after infection;
Figure 7D-F). In addition, no statistically significant differences
were detected in the fold change of the absolute number of CD4⫹
T cells and number of naive CD4⫹ T cells (supplemental Figure
1A-F). Besides a typical lentiviral infection-induced lymphopenia
around the peak viremia, which was observed in both groups of
animals, the number of CD4⫹ T cells (total number and number of
memory cells, naive cells, and Ki-67⫹ cells) showed little change
compared to levels seen before infection.
Consistent with the similarity in CD4 T lymphocytes between
the 2 groups, the disease-free survival was not affected during a
follow-up period of 1 year in the CD8⫹ and CD20⫹ lymphocytedepleted AGMs. Regardless of the treatment, none of the 12
animals investigated showed any signs of an AIDS-like disease.
These observations furthermore underline the robustness of the
AGM immune system toward SIV infection, even under harsh
treatment such as simultaneous CD8⫹ and CD20⫹ lymphocyte
depletion.
Discussion
This study demonstrates that transient inhibition of CD8⫹ T cell–
and B cell–mediated adaptive immune responses during primary
SIV infection of sabaeus AGMs did not lead to the induction of an
AIDS-like disease. However, the transient absence of CD8⫹
lymphocytes did result in a delay of the initial viral containment.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3076
ZAHN et al
BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
Figure 7. CD8ⴙ and CD20ⴙ lymphocyte depletion in SIVinfected AGMs did marginally affect the number of memory
CD4ⴙ T cells and Ki-67ⴙ memory CD4ⴙ T cells in peripheral
blood. CD4⫹ T cells of control Ab-treated AGMs (A-D) and CD8⫹
and CD20⫹ lymphocyte-depleted AGMs (B-E) were stained with
anti-CD28 and anti-CD95 to determine naive (CD95⫺) and memory
(CD95⫹) CD4⫹ T-cell subsets. The fold change in memory CD4⫹
T-cell numbers (A-C) and the fold change in Ki-67⫹ memory CD4⫹
T-cell numbers (D-E) were calculated for each time point compared with the median of 3 preantibody injection time points.
(C) The median fold change in memory CD4⫹ T cells from the
6 control Ab-treated AGMs (solid line, filled symbols) and the
6 CD8⫹ and CD20⫹ lymphocyte-depleted AGMs (solid line, open
symbols) are shown. (F) The median fold change in Ki-67⫹
memory CD4⫹ T cells from the 6 control Ab-treated AGMs (solid
line, filled symbols) and the 6 CD8⫹ and CD20⫹ lymphocytedepleted AGMs (solid line, open symbols) are shown. *Single time
point with a significant difference (Mann-Whitney test) between
the control group and the antibody-treated group of animals. No
significant differences were detected between the control and
antibody-treated groups at any of the other time points. p.i.
indicates postinfection.
The reappearance of CD8⫹ lymphocytes and the generation of a
high frequency of SIV-specific CD8⫹ T cells were associated with a
reduction of viremia to set-point levels. In contrast, the delay in
humoral immune responses in B cell–depleted animals did not
correlate with viremia.
A number of investigations have shown that natural hosts are
capable of eliciting SIV-specific adaptive immune responses and
that these responses are of a similar or slightly lower magnitude
compared with non-natural hosts infected with SIV.27-30,39-41 However, there is also accumulating evidence suggesting that natural
hosts may use evolutionary adaptations to remain disease-free.42 To
determine the role of adaptive immune responses in the maintenance of a disease-free course of infection, we used a combined
treatment with both anti-CD8 and anti-CD20 antibodies to suppress
the generation of adaptive immune responses in sabaeus AGMs.
Even under these extreme conditions, the lymphocyte-depleted
AGMs showed only a temporary elevation in viremia during
primary infection. Similar to the control group, the lymphocytedepleted AGMs showed only a brief decline in total CD4⫹ T cells
in peripheral blood. As expected in AIDS virus infections, CCR5⫹
CD4⫹ T cells (data not shown) and memory CD4⫹ T cells showed
some decline after infection, albeit we could not detect a difference
between both groups of AGMs studied. None of these changes had
any impact on the course of SIV infection; both groups of AGMs
remained disease-free.
This observation is in sharp contrast to observations made in
CD8⫹ or CD20⫹ lymphocyte depletion studies in pathogenic SIV
infection models.5,15,16,43 CD8⫹ lymphocyte depletion during primary viremia in rhesus monkeys resulted in an extremely high
viremia (in the range of 108 copies viral RNA/mL plasma). As a
consequence of CD8⫹ lymphocyte depletion during primary infection, CD8⫹ T-cell responses, B-cell responses, and CD4⫹ T-cell
responses were significantly impaired or did not develop, and
survival was drastically decreased.36,44 Similarly, CD8⫹ and CD20⫹
lymphocyte depletion during primary SIVagm infection of pigtailed
macaques resulted in increased viremia and accelerated disease.45
Studies of rhesus monkeys during primary SIV infection showed
that humoral immune responses do not contribute to early viral
containment. In these studies, the level of primary viremia was
indistinguishable between B cell–depleted and control animals.15,16,45 In
the present study, it is difficult to assess the role of humoral immune
responses as we impaired the generation of both cellular and humoral
immune responses. However, correlative evidence in the control group
suggests that humoral immune responses do not contribute to viral
containment in primary SIV infection of sabaeus AGMs and to a
disease-free course of infection. Thus, a stable set-point viremia was
established at week 5 after infection at a time when NAbs were still
undetectable in most animals.
In contrast to primary infection, some investigators have
recently suggested that humoral immune responses may contribute to viral control in chronic SIV viremia.16 In these studies, a
significantly reduced survival was observed in long-term B cell–
depleted SIV-infected rhesus monkeys. In the present study in
sabaeus AGMs and in recent investigations in vervet AGMs,45
the appearance of NAbs did not affect the magnitude of plasma
viral RNA in lymphocyte-depleted AGMs. In addition, B-cell
depletion did not affect survival. Also, recent B-cell depletion
experiments in SIV-infected sabaeus AGMs performed by other
investigators confirmed our findings: inhibition of humoral
immune responses in AGMs did not result in any adverse side
effects or the induction of an AIDS-like disease.46 Still, we
cannot formally rule out that more aggressive and longer-term
inhibition of humoral immune responses may result in a
different outcome. However, in general, there is little evidence
in HIV infection of humans and SIV infection of nonhuman
primates that NAbs significantly reduce the level of viremia.
There is evidence that the decreased survival after long-term
depletion of B cells in SIV-infected rhesus monkeys may be the
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BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
result of an indirect effect. We observed a lower magnitude of
CD8⫹ T-cell responses during chronic SIV infection in longterm B cell–depleted SIV-infected rhesus monkeys (M. de la
Rosa, P. Chugh, V. Evans, S. Finstad, S. Westmoreland, A.C.,
Michael Piatak, J. D. Lifson, D. C. Montefiori, and J.E.S.,
unpublished observation, January 2008).
CD8⫹ lymphocyte depletion experiments in natural hosts and
non-natural hosts suggest that CD8⫹ T-cell responses participate
in viral control. Interestingly, significant differences were seen
between rhesus macaques and sabaeus AGMs when CD8⫹
lymphocytes reappeared. In rhesus monkeys, the recovery of
CD8⫹ T cells and the generation of SIV-specific responses were
highly suppressed.36,44 In contrast, SIV-infected sabaeus AGMs
that usually exhibit only relatively low CD8⫹ T-cell responses
showed a dramatic increase in SIV-specific responses when
CD8⫹ T cells reappeared. The transient prolongation of primary
viremia in CD8⫹ lymphocyte-depleted AGMs may have crossed
a threshold necessary to induce a vigorous expansion of
SIV-specific CD8⫹ T cells. Observations made in the CD8⫹
lymphocyte-depleted AGM no. 364 support this notion. CD8⫹
lymphocytes were only inefficiently and briefly depleted in this
animal, and replenishment was not associated with a high
number of Env-specific CD8⫹ T cells. The magnitude of
SIV-specific CD8⫹ T-cell responses seen after the reappearance
of CD8⫹ lymphocytes in most of the CD8⫹ lymphocytedepleted AGMs also refutes the notion that natural hosts are
only capable of relatively subdued immune responses to SIV.
Our data clearly show that sabaeus AGMs are capable of
generating robust SIV-specific CD8⫹ T-cell responses. Our
findings also suggest that the presence of viremia does not
abrogate the function of virus-specific CD8⫹ T-cell responses in
AGMs. Thus, CD8⫹ lymphocyte recovery in AGMs (in contrast
to rhesus monkeys36,44) may be associated with an appropriate
response sufficient to reduce the viremia to an “asymptomatic”
set-point level.
In addition to the effect of CD8⫹ lymphocyte depletion on SIV
viremia, we measured a significantly increased level of cytomegalovirus
(CMV) viremia during lymphocyte depletion (supplemental Figure 2).
This indicates a critical role of these cells in controlling CMV infection
in AGMs. This finding is in agreement with previous observations in
other nonhuman primates, humans, and mice.31,47-49 Interestingly, we
did not observe a generalized immune activation despite the reactivation
of CMV or the massive expansion of CD8⫹ T cells after the reappearance of these cells, indicating an inherent stability and regulation of the
immune system in these animals.
Our observations suggest that CD8⫹ T cells are at least partially
involved in viral containment in AGMs as the vigorous expansion of
SIV-specific CD8⫹ T cells coincided with the partial containment of
viremia. We cannot rule out that NK cells participate in the partial
control of the viremia as they also are depleted by the anti-CD8␣
antibody and reappear coincident with CD8⫹ T cells. In addition, the
ultimate role and possible limitations of CD8⫹ T-cell responses in
AGMs are still unknown. We currently have no data on the breadth of
the SIV-specific immune responses beyond Env and Gag responses.
However, it was remarkable that the primary CD8⫹ T-cell response was
mainly directed against Env (similar to SIV-specific antibody responses). The eventual decline of these Env responses to levels similar
to those seen for Gag responses suggests that SIVsab may also undergo
sequence mutations resulting in immune escape. Limited data from
another natural host, SIVsm-infected sooty mangabeys, have suggested
that escape from cellular immune responses does occur.50 However, this
area requires further investigation.
ROLE OF SIV-SPECIFIC IMMUNE RESPONSES IN AGM
3077
It is tempting to generalize the observations made here to other
natural host species. However, the mechanisms of protection used by
natural hosts to evade pathogenic consequences of SIV infection are
probably the result of complex evolutionary adaptations and thus may
differ considerably between the 40 different monkey and ape natural
host species. Therefore, future efforts should include additional natural
host species to determine the breadth of immune and nonimmune
protection mechanisms used by natural hosts. The study of natural hosts
gives researchers the unique opportunity to study AIDS virus-infected
animals that do not develop disease despite relatively high viral loads. In
contrast, less pathogenic AIDS virus infections of non-natural hosts (ie,
HIV-2 in humans or SHIV89.6 in rhesus macaques) are always
associated with a relatively low-level set-point viremia.
In conclusion, this study suggests that CD8⫹ lymphocytes
contribute to partial viral containment during primary SIV
infection in AGMs. However, the temporal inhibition of adaptive immune responses had no apparent effect on the SIVinfected AGMs. It would be reassuring for current AIDS vaccine
efforts if adaptive immune responses also contribute to the
disease-free course of infection observed in natural hosts.
However, it is still unclear whether these responses are at all
necessary for preventing outbreak of overt signs of SIVmediated disease in natural hosts. Future in vivo manipulations
aiming to achieve longer-lasting suppression of adaptive immune responses and/or induction of chronic immune activation
will be required to address these questions. A more precise
picture of the breadth of mechanisms used to prevent disease in
natural hosts of lentivirus infection may provide important
insights for the generation of a more successful AIDS vaccine
and/or development of novel treatment modalities.
Acknowledgments
This work was supported by the NIH (grants RR000168, New
England Primate Research Center; AI43890, A.K.; AI30034,
D.C.M.; AI065335, J.E.S.), NIAID Center for HIV/AIDS Vaccine
Immunology (grant AI067854, J.E.S., N.L.L.), and the Harvard
Medical School Center for AIDS Research (grant AI060354).
Reagents used in this work were provided by the NIH Nonhuman
Primate Reagent Resource (grants AI040101 and RR016001).
Authorship
Contribution: R.C.Z., V.M.H., J.S.A., and J.E.S. conceived and
designed the experiment; M.D.R., M.L., H.T., B.K.-S., H.B., R.W.,
and S.P. performed the experiment; R.C.Z., N.L.L., A.K., D.C.M.,
V.M.H., J.S.A., and J.E.S. analyzed the data; R.C.Z. and J.E.S.
wrote the paper; and A.C. performed the experimental work with
nonhuman primates.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
Correspondence: Jörn E. Schmitz, Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Center for Life Science,
E/CLS 1037, 330 Brookline Ave, Boston, MA 02215; e-mail:
[email protected].
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3078
BLOOD, 15 APRIL 2010 䡠 VOLUME 115, NUMBER 15
ZAHN et al
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2010 115: 3070-3078
doi:10.1182/blood-2009-10-245225 originally published
online February 10, 2010
Suppression of adaptive immune responses during primary SIV
infection of sabaeus African green monkeys delays partial containment
of viremia but does not induce disease
Roland C. Zahn, Melisa D. Rett, Ming Li, Haili Tang, Birgit Korioth-Schmitz, Harikrishnan
Balachandran, Robert White, Sarah Pryputniewicz, Norman L. Letvin, Amitinder Kaur, David C.
Montefiori, Angela Carville, Vanessa M. Hirsch, Jonathan S. Allan and Jörn E. Schmitz
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