1133
Decline of Simian Immunodeficiency Virus (SIV)–Specific Cytotoxic T
Lymphocytes in the Peripheral Blood of Long-Term Nonprogressing Macaques
Infected with SIVmac32H-J5
1
Institute of Virology, Erasmus University, Rotterdam, and 2Central
Animal Laboratory, National Institute of Public Health
and the Environment, Bilthoven, The Netherlands; 3Paul-Ehrlich
Institute, Langen, Germany; 4UMR103, CNRS-Biomerieux,
Ens de Lyon, Lyon, France
The evolution of simian immunodeficiency virus (SIV)–specific cytotoxic T lymphocyte
precursors (CTLps) and their relationship with virus replication were studied in SIV-infected
macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain
reaction–detectable provirus in peripheral blood. Although proviral DNA persisted in the
spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout
the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings
were associated with the detection of CTLps directed against both structural and regulatory
SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic
stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to
reinfection. These findings bear implications for vaccination strategies aimed at inducing
protective CTLs against lentiviruses.
Although the role of cytotoxic T lymphocyte (CTL) responses in the host defense against lentiviruses has not yet been
firmly established, several observations indirectly support the
view that CTLs are important correlates of protection against
human immunodeficiency virus (HIV) infection and disease, as
reviewed elsewhere [1]. CTLs against HIV antigens have been
detected in persons exposed to infectious body fluids but lacking
in evidence of infection [1], suggesting, although not proving,
that virus clearance by CTLs is indeed possible. A temporal
association has also been described between the development
of HIV-specific CTLs and the containment of primary viremia
[1]. Furthermore, during the early stage of infection, plasma
RNA loads show an inverse correlation with the levels of Envspecific CTLs [2], whereas the detection of Rev-specific CTL
precursors (CTLps) predicts a subsequent slow rate of disease
progression [3].
Received 9 February 1999; revised 11 June 1999; electronically published
8 September 1999.
Financial support: Dutch Health Research Council, Dutch AIDS Foundation (grant g1-027).
a
Present address: Department of Virology, Division of Pathology and
Communicable Diseases, Royal Free Campus, University College, UK
(geretti@rfhsm.ac.uk).
Reprints or correspondence: Albert D. M. E. Osterhaus, Institute of Virology, Erasmus University, Dr. Molewaterplein 50, P.O. Box 1738, 3000
DR Rotterdam, Netherlands (osterhaus@viro.fgg.eur.nl).
The Journal of Infectious Diseases 1999; 180:1133–41
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/1999/18004-0026$02.00
After the early phase of infection, the relationship between
HIV-specific CTLs and the rates of virus replication and disease
progression appears to be less than clear. Earlier studies have
proposed that high levels of CTL activity are associated with
the maintenance of the asymptomatic status, whereas CTL responses decline with disease progression [4]. Consistent with
these observations, an inverse correlation has been found between plasma RNA load and the levels of CD81 T lymphocytes
that recognize HIV Gag or Pol peptides in the context of major
histocompatibility complex (MHC) class I tetrameric complexes [5]. Nevertheless, strong CTL responses have occasionally been detected in persons with advanced disease [6], and,
conversely, low levels of CTL activity have been observed in
persons with slow rates of disease progression [7]. In fact, declining CTL responses to HIV antigens have been observed in
a long-term nonprogressor carrying a defective HIV provirus
and lacking detectable viral RNA expression [8].
Macaque simian immunodeficiency virus (SIVmac) shares
with HIV many biological and structural features, a similar
genomic organization, and extensive genetic homology. The virus establishes a persistent infection in macaques and induces
an immunodeficiency syndrome closely resembling human
AIDS [9]. Simian AIDS, however, runs a more rapid course,
as most animals develop overt disease within 1–4 years of infection. Nevertheless, animals of the same species and origin
show a considerable variation in the rate of disease progression;
some develop symptoms and die within a few months, whereas
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Anna-Maria Geretti,1,a Ellen G. J. Hulskotte,1
Marlinda E. M. Dings,1 Carel A. van Baalen,1
Geert van Amerongen,2 Stephen G. Norley,3
Patrick Boers,1 Rob Gruters,1,4
and Albert D. M. E. Osterhaus1
1134
Geretti et al.
Materials and Methods
Macaques. Eight cynomolgus macaques were infected intravenously with either 50 (primary infection) or 200 (reinfection)
monkey median infectious doses of cell-free SIVmac32H-J5 [10].
Monkeys K77, K80, K83, and K88 had been previously immunized
with an SIV subunit vaccine but became infected upon challenge
[10]. Observation lasted for at least 13 months (K73 and K79) and
up to 22 months (K66, K71, K77, K80 and K88). Monkey K83
was euthanized 18 months after infection upon the development
of symptomatic disease with diarrhea and weight loss. Macaques
were anesthetized with ketamine for blood sampling and lymph
node biopsies. Spleens were removed at the end of observation.
Mononuclear cell preparation. Lymph nodes and spleen, in
complete culture medium (RPMI 1640 with 100 U/mL penicillin,
100 mg/mL streptomycin, 1025 M b-mercapto ethanol, and 2 mM
L-glutamine) containing 10% fetal calf serum (FCS), were teased
with forceps to place cells in suspension and were filtered through
a 112-mm-pore-size nylon mesh. Peripheral blood, lymph node, and
splenic mononuclear cells (PBMC, LNMC, and SPMC) were isolated by density-gradient centrifugation [10] and were either used
immediately or cryopreserved. Herpesvirus papiotransformed
B–lymphoblastoid cell lines (B-LCL) were established from PBMC
by incubation with cell-free supernatant from cell line S594 [10].
Subset-cell separation was done with antibody-coated magnetic
beads (Dynabeads M-450; Dynal, Oslo, Norway), as described elsewhere [11]. For flow cytometry, cells in PBS with 1% bovine serum
albumin were incubated for 30 min with anti-CD2 (Leu-5b-FITC;
Beckton Dickinson, Mountain View, CA) and anti-CD8 (Leu-2aPE; Beckton Dickinson) or anti-CD4 (OKT4-FITC; Ortho Diagnostic System, Raritan, NJ) monoclonal antibodies, washed twice,
fixed in 1% paraformaldehyde (PFA), and analyzed with a FACscan
flow cytometer (Beckton Dickinson). Rates of change of CD41 cell
percentages were obtained by linear regression.
Virus detection. Virus load was determined in serial dilutions
of freshly isolated PBMC, LNMC, SPMC and in their CD8-depleted fractions, cocultured with the human T-cell line C8166, as
described elsewhere [10]; the lowest dilution contained 106 cells.
SIV p26 core antigenemia was assayed by antigen-capture ELISA
(Coulter SIV Core Antigen Assay; Coulter Electronics, Mijdrecht,
Netherlands). A nested polymerase chain reaction (PCR), adapted
from Chen et al. [12], was used to amplify a 500-bp fragment of
SIV Gag. Briefly, DNA was extracted [13] from 106 freshly isolated
PBMC, LNMC, and SPMC CD4–enriched fractions, and from the
same cells after coculture with C8166 cells. After heparinase treatment, DNA was added to a solution containing PCR buffer
(50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100),
MgCl2 (1.5 mM), dNTP (0.2 mM), Taq polymerase (1 U), and
primers (31 pM). Two sets of primers were used, for outer (5′CATTCACGCAGAAGAGAAAG;
3′-GCATTTTGAATCA′
GCAGTG) and inner (5 -ACAAGTAGACCAACAGCACC; 3′TGAAATGGCTCTTTTGGCC) amplification, respectively. PCR
conditions were 1 min, 10 s at 947C; 1 min, 30 s at 507C; and 1
min at 727C for 25 and 30 cycles, with a 10-min final extension.
The nested SIV Gag PCR was highly sensitive, and the control
plasmid could be diluted 5 log without loss of signal. The higher
limit of sensitivity was 1 in 10,000 infected cells. Sequence analyses
were conducted both directly and after cloning.
Antibody detection. Anti-Env antibodies were measured by indirect ELISA [10]. Virus-neutralizing antibodies were measured in
a yield reduction assay. Briefly, 1 : 50 serum dilutions (20 mL/well)
were incubated for 30 min at 377C with 20 mL of serially diluted
(3-fold) SIVmac32H in 8 replicates per dilution. C8166 cells
(1.33 3 10 4/mL) were added to each well in 150 mL. After incubation for 7 days, cells were transferred to poly-L-lysine–coated
plates and fixed in methanol. Infected cells were detected by immunoperoxidase staining [10]. The yield reduction was calculated
as the ratio of virus titer (TCID50/mL) in the absence of serum to
the titer in the presence of the sample serum.
Generation of effector cells. Optimal conditions for the detection of SIV-specific CTLps by limiting dilution assay (LDA) were
established as described elsewhere [11]. Effector cells were expanded
by two 1-week cycles of stimulation with autologous PFA-fixed BLCL (104/well) infected with recombinant vaccinia vectors (rVVs)
expressing SIV antigens. The optimal numbers and ranges of dilutions and numbers of replicate wells were predetermined for each
monkey in a pilot experiment; cultures included at least 4 and up
to 8 cell dilutions; each dilution included at least 24 and up to 48
replicate wells. Cultures also contained autologous irradiated (2500
rad) feeder PBMC (104/well), recombinant interleukin-2 (rIL-2, 10
U/mL) from day 3, and 10% supernatant from concanavalin
A–stimulated blasts from day 7. The following rVVs were used:
SIVmac32H Env [10] and Gag p55 (provided by Prof. A. McMichael, Institute of Molecular Medicine, Oxford, UK); SIVmac32H-J5 Nef [10] and Rev (vv9005; MRC AIDS–Directed Programme, Potters Bar, South Mills, UK); SIVmac-BK28 Pol
(ADP263; MRC AIDS–Directed Programme); and SIVmac251 Tat
(TG4174; Transgene, Strasbourg, France). The vector 186-poly
(Transgene), containing a polycloning site without insert, was used
as a control. For peptide-specific stimulation, effector cells were
cultured for 14 days with autologous irradiated (5000 rad) peptidepulsed B-LCL (104/well), autologous irradiated feeder PBMC (104/
well), and rIL-2 (10 U/mL) from day 4. A 9-mer peptide designated
p26A.5 [14], covering amino acids 242–250 of p26 (SVDEQIQWM)
was synthesized at the European Veterinary Laboratory (Woerden,
Netherlands).
Cytotoxicity assay and calculation of CTLp frequencies. Cytotoxicity was measured in standard 51Cr-release assays, as described elsewhere [11, 14]. Target cells were autologous and MHC
class I–mismatched allogeneic B-LCL incubated overnight with
either SIV rVVs, vaccinia virus control, peptide p26.A5, or medium
alone. Individual LDA wells were considered positive when lysis
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others remain asymptomatic for a few years, thus resembling
long-term nonprogressors with HIV infection.
Because of its similarities with HIV infection of humans,
SIVmac offers a unique model to explore the relationship between antiviral immune responses and the long-term containment of lentiviral infections. Here, we investigated the evolution
of SIV-specific CTLps in macaques infected intravenously with
a molecular clone of SIVmac and followed up prospectively for
up to 22 months. We identified 3 animals that, in coincidence
with effective virus containment, progressively lost their CTL
response against SIV. This finding bears implications for the
design and evaluation of vaccines against SIV or HIV infection.
JID 1999;180 (October)
JID 1999;180 (October)
Kinetics of SIV-specific CTL
of targets expressing SIV antigens exceeded that of control targets
by 10%, if the latter was !10%, or by 20%, if the latter was 110%.
Frequencies were estimated by the maximum-likelihood method
with a software package that included a x2 goodness-of-fit test
statistic [15]. Only estimates with test statistics !10 were accepted.
Frequencies were normalized to the number of CTLp/106 PBMC,
LNMC, or SPMC. Rates of change of CTLp frequencies were
calculated by linear regression.
Prospective virological and clinical evaluation of 8 SIV-infected
macaques. Eight cynomolgus macaques were infected intravenously with SIVmac32H-J5, a pathogenic molecular clone
derived from SIVmac. In monkey K71, PBMC-associated virus
load and p26 antigenemia showed a peak at week 2 after infection (11000 infected cells/106 PBMC and 1.10 ng/mL, respectively), followed by a rapid decline. Repeated attempts to
isolate virus from PBMC and their CD8-depleted fractions (to
remove potential virus-suppressive factors) were unsuccessful
between 3 and 6 months after infection (table 1). However,
during this time, proviral DNA was detected in PBMC by
nested Gag-PCR. Sequence analyses showed that PBMC carried a 520-bp Gag fragment identical to that of the J5 clone
used for challenge (not shown). Between 12 and 20 months
after infection, virus isolation remained unsuccessful. In addition, repeated attempts failed to detect proviral DNA in
PBMC, their CD4-enriched fractions (to increase sensitivity),
and the same cells after coculture with C8166 cells. Identical
observations were made with monkeys K77 and K80 between
12 and 22 months after infection. In contrast, virus was detected
in PBMC of another group of 5 monkeys (K66, K73, K79,
K83, and K88) for the entire length of observation (minimum
13 months). Seven of the 8 monkeys remained asymptomatic
during their observation periods. Monkey K83 developed
symptomatic disease 18 months after infection. The percentages
of circulating CD41 cells (table 1) were stable over time in
monkeys K71, K77, and K80 but declined significantly (P !
.05) in the other 5 monkeys.
Specificity of circulating CTLps. The optimal conditions
for the detection of SIV-specific CTLps in cynomolgus macaques were established as described elsewhere [11]. The frequencies of Env-, Gag-, Pol-, Tat-, Nef- and Rev-specific CTLps
were measured by antigen-specific stimulation of PBMC collected from each macaque before and after infection. Frequencies in naive PBMC were always !2/106 PBMC (not shown).
After infection (table 1), all animals had CTLps against Gag
and Tat, and most also had CTLps against Env (5/7), Nef (4/
7), or Pol (4/7). Only 3 animals, K71, K77, and K80, showed
Rev-specific CTLps at frequencies 15/106 PBMC in at least 2
independent experiments. Consistent with previous data reviewed elsewhere [16], even at the peak of response, CTLp
frequencies were often !15/106 PBMC, but estimates up to 121/
106 PBMC were found in monkeys K71, K80, and K88.
Kinetics of SIV-specific CTLps. CTLp frequencies were
compared in PBMC collected at multiple times during up to
22 months of observation (figure 1). Samples obtained from
each macaque at different times were tested simultaneously by
use of identical culture and assay conditions, including identical
stimulator cells, rIL-2, feeder cells, and 51Cr-labeled targets.
Monkey K83 developed a relatively low frequency (up to 13/
106 PBMC) of multispecific CTLps during the first 6 months
of infection and lost detectable CTLps against Env, Tat, and
Nef during the subsequent 6 months but maintained anti-Gag
CTLps 18 months after infection, the time of onset of symptomatic disease. CTLp frequencies remained either stable or
increased over time in monkeys K66, K79, and K88. In contrast, CTLps of monkeys K71, K77, and K80 peaked within
the first 7 months of infection but declined significantly thereafter. Although Env- and Gag-specific CTLps showed the most
marked decline (figure 2), 20–22 months after infection, all SIVspecific CTLps in the 3 monkeys had declined to levels significantly lower than those measured at 4 months.
Table 1.
Prospective virological and immunological evaluation of simian immunodeficiency virus–infected
macaques.
a
b
VC/PCR (mo.)
CTLp specificity
Monkey
1–2
3
4–6
12
18–20
Env
Gag
Pol
Tat
Nef
Rev
K83
K66
K73
K79
K88
K71
K77
K80
1
1/1
1/1
1/1
1
1/1
1
1
1
2/1
1
1
1
2/1
2/1
1
1
2/1
1
1
1
2/1
1
1
1
2/1
1
1
1
2/2
2/2
2/2
1
2/1
ND
ND
1
2/2
2/2
2/2
1
1
ND
(1)
111
2
1
111
1
11
1
1
111
111
1
1
1
1
ND
1
2
2
2
11
1
11
ND
1
111
11
1
1
1
(1)
ND
11
(1)
2
1
11
2
(1)
2
2
2
1
11
1
a
c
CD4 slope
—0.8
2 0.8
21.1
20.7
20.3
0
0
0
5
5
5
5
5
0.2
0.2
0.4
0.3
0.1
(.001)
(.0004)
(.004)
(.004)
(.004)
Virus culture (VC) was done with peripheral blood mononuclear cells (PBMC) and their CD8-depleted fractions; polymerase
chain reaction (PCR) was done with DNA from both freshly isolated and cultured PBMC and their CD4-enriched fractions; 1,
VC positive; 1/1, VC positive, PCR positive; 2/1, VC negative, PCR positive; 2/2, VC negative, PCR negative.
b
Quantitative data indicate highest cytotoxic T lymphocyte precursor (CTLp) frequency measured during whole observation
period and reproduced in at least 2 independent experiments; 2, !2; (1), 2–4; 1, 5–14; 11, 15–50; 111, 150 CTLp/106 PBMC.
c
Rates of change with SE (and P values) of CD41 cell percentages/month determined from 6 to 9 measurements per monkey
(mean 8.1). ND, not done.
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Results
1135
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Geretti et al.
JID 1999;180 (October)
SIV protein- and epitope-specific CTLps of monkey K71.
To confirm and extend the previous observations, detailed studies were conducted with monkey K71, 1 of the animals showing
good virus containment and whose CTLp frequencies were initially 115/106 PBMC. By use of both polyclonal and clonal
CTL populations, we have shown previously that CD81 and
MHC class I–restricted CTLs of this monkey recognized an
immunodominant 9-mer epitope in Gag, designated p26.A5,
spanning amino acids 242–250 (SVDEQIQWM) of p26 [14].
Here, we compared the frequencies and kinetics of Gag-specific
CTLps with those of p26.A5-specific CTLps. After PBMC stimulation with Gag rVV, 4 aliquots from individual LDA wells
were tested in split-well assays against Gag rVV, peptide
p26.A5, and 2 control targets. CTLps against p26.A5 represented 77% of Gag-specific CTLps early after infection but only
31%–39% between 4 and 7 months after infection, indicating
that Gag recognition was broader at the peak of response (table
2).
The frequencies of Gag-specific CTLps, after reaching a plateau, decreased more rapidly than those of p26.A5-specific
CTLps. The rates of decline from 4 months after infection were
24.2 5 0.6/month (P = .003) and 21.1 5 0.3/month (P = .01),
for Gag- and p26.A5-specific CTLps, respectively. As a result,
between 12 and 20 months after infection, CTLps against
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Figure 1. Kinetics of simian immunodeficiency virus (SIV)–specific cytotoxic T-lymphocyte precursors (CTLps) in SIV-infected macaques.
Effector cells were expanded with recombinant vaccinia vectors expressing SIV Env, Gag, Pol, Tat, Nef, or Rev. Frequencies were normalized to
number of CTLp/106 peripheral blood mononuclear cells (PBMC).
JID 1999;180 (October)
Kinetics of SIV-specific CTL
p26.A5 constituted 90%–100% of Gag-specific CTLps. The reproducibility of CTLp frequencies was confirmed in independent replicate experiments for each time point (table 2). In
addition, the frequencies of p26.A5-specific CTLps measured
after stimulation with Gag rVV were reproduced in PBMC
expanded by stimulation with peptide p26.A5 (table 2).
Rechallenge of monkey K71. Twenty months after primary
infection, monkey K71 was rechallenged intravenously with a
high dose of SIVmac32H-J5. Two naive monkeys were infected
as controls. Consistent with previous findings [10], a burst of
virus replication was observed in the 2 naive monkeys between
2 and 5 weeks after infection (not shown). In monkey K71, a
transient and modest increase in virus burden was observed at
week 6, coinciding with a transient decrease of circulating CD41
cells. The numbers and percentages of circulating CD81 cells
and Env-specific antibody titers showed a significant increase
(figure 3). Virus-neutralizing antibody titers, however, did not
change significantly.
CTLp frequencies were measured simultaneously in all
PBMC samples collected during 10 weeks of observation by
use of identical culture and assay conditions. Gag-specific
CTLps showed a progressive increase after reinfection, and by
week 4 their frequency (22/106 PBMC; range, 15–28; x 2 = 1)
was similar to that measured at week 4 after primary infection
(27/106 PBMC; range, 17–37; x 2 = 4). In the same cultures tested
by split-well assay, p26.A5-specific CTLps also showed a rapid
but less pronounced increase after reinfection and by week 10
constituted 50% of circulating Gag-specific CTLps.
Comparison of peripheral blood, lymph nodes, and spleen 22
months after infection. After 22 months of infection, virus
isolation and PCR data from peripheral blood of 5 monkeys
were compared with those from their spleen and axillary, in-
guinal, and mesenteric lymph nodes (table 3). By culture, virus
was recovered from the blood, lymph nodes, and spleen of
monkeys K88 and K66. By use of nested PCR, provirus DNA
was detected in the lymph nodes and spleen of all 5 monkeys,
including the 3 monkeys (K71, K77, and K80) that lacked
detectable virus in peripheral blood. However, in these 3 monkeys, cultures of whole mononuclear cells and their CD8-depleted fractions yielded a virus load either below (lymph nodes)
or barely above (spleen) detection levels.
In the reinfected monkey K71, virus load was either similar
(spleen) or lower (lymph nodes) than that detected in superficial
lymph nodes at weeks 12 and 23 after primary infection (figure
4). CTLps against Gag and p26.A5 were measured simultaneously in cells from the lymph nodes, spleen, and peripheral
blood. In lymph nodes, all anti-Gag CTLps also recognized
the p26.A5 epitope (figure 4). Their frequency (up to 12/106
mononuclear cells) was lower than that in blood (up to 40/106
PBMC) but similar to that detected in superficial lymph nodes
at weeks 12 and 23 after primary infection (up to 13/106 mononuclear cells). Higher Gag-specific CTLp frequencies were
found in the spleen (up to 30/106 mononuclear cells), and, as
in the blood, these included a proportion of CTLps directed
against targets other than p26.A5. These observations were
confirmed after adjustment of CTLp frequencies to the initial
proportion of CD81 T cells in PBMC (50%), lymph nodes
(36%–40%), and spleen (47%).
Discussion
In the present study, we followed the evolution of SIV-specific
CTLps in macaques infected intravenously with a molecular
clone of SIVmac. We found that effective virus containment,
as indicated by the loss of detectable virus in peripheral blood
Table 2. Gag- and p26.A5-specific cytotoxic T-lymphocyte precursors
of monkey K71.
a
CTLp specificity
SIV Gag
p26.A5
Gag-stimulation
Months
1.4
4
6
7
10
12
15
20
a
Exp 1
30
58
62
64
41
17
13
3
(21–29)
(44–73)
(45–79)
(48–80)
(30–53)
(10–23)
(9–16)
(1–6)
Exp 2
ND
56 (42–70)
60 (45–74)
ND
ND
20 (13–26)
ND
3 (0.4–6)
Gagstimulation
p26.A5stimulation
23
18
21
25
20
16
12
3
24 (16–32)
ND
ND
23 (17–29)
17 (11–23)
18 (12–24)
ND
3 (0.4–6)
(16–30)
(12–25)
(15–28)
(17–33)
(13–27)
(9–22)
(9–16)
(1–6)
Effector cells were generated by stimulation of peripheral blood mononuclear
cells (PBMC) with either simian immunodeficiency virus (SIV) Gag recombinant
vaccinia vector (rVV) or peptide p26.A5, as indicated. After two 1-week cycles
of stimulation, 4 aliquots from each well of limiting dilution cultures were tested
for cytotoxicity against autologous target B-LCL infected either with Gag rVV
or with vaccinia virus control, sensitized with peptide p26.A5, or incubated with
medium alone. Frequencies (with 95% confidence intervals) were normalized to
number of cytotoxic T lymphocyte precursors/106 PBMC. ND, not done.
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Figure 2. Decline of simian immunodeficiency virus (SIV)–specific
cytotoxic T lymphocyte precursors (CTLps) in monkeys K71, K77,
and K80. CTLp frequencies were compared at 4, 12, and 20 (K71) or
22 (K77 and K80) months after infection. A, For each monkey, total
CTLps were obtained by summing individual Env- Gag-, Pol-, Tat-,
and Nef-specific CTLps. B, For each SIV protein, total CTLps were
obtained by summing CTLp frequencies of individual macaques.
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Geretti et al.
JID 1999;180 (October)
and detection of low-level virus reservoirs in lymphoid tissues,
resulted in declining CTLp frequencies in circulation.
For the detection of SIV-specific CTLps, cells under limiting
dilution conditions were restimulated with rVVs expressing SIV
antigens. In previous studies of the same macaques, this approach led to the expansion of CTL with a CD81 and MHC
class I–restricted phenotype [11, 14]. We have extensively investigated the conditions for the detection of SIV-specific
CTLps in cynomolgus macaques [11] and have found that a
reproducible assessment can be obtained by tailoring the LDA
settings to individual macaques and by rejecting CTLp frequencies with x2-test statistics 110.
CTLps detected by LDA are believed to be correlated with
antiviral T-cell memory in vivo [17]. Recently, quantitation of
CD81 T cells by fluorescent staining with tetrameric MHC-
peptide complexes has suggested that LDA may underestimate
by ∼10-fold the actual size of the memory CTL pool. It is
noteworthy, however, that despite this potential underestimation, the kinetics and duration of memory CTL responses determined by LDA have been shown to be correct [18]. Our
kinetics studies identified 3 patterns of response in SIV-infected
macaques. In asymptomatic macaques with persistent virus in
PBMC (i.e., K66, K79, and K88; table 1), CTLps were stable
or even increased over time (figure 1). In contrast, overall
CTLps declined before the onset of symptoms in the monkey
that developed overt disease (i.e., K83; figure 1). Although the
latter finding agrees with observations made in HIV-infected
humans and supports a role for CTLs in preventing disease
progression, it is noteworthy that Gag-specific CTLps were
maintained in monkey K83 at the time of progression.
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Figure 3. Primary (left) and secondary (right) intravenous simian immunodeficiency virus infection of monkey K71. A, Virus load, measured
in peripheral blood mononuclear cells (PBMC) and their CD8-depleted fractions, was normalized to number of infected cells/106 PBMC. Polymerase
chain reaction (PCR) was done with DNA from both freshly isolated and cultured PBMC and their CD4-enriched fractions. M, PCR negative;
m, PCR positive. B, Percentages (solid lines) and absolute numbers (dotted lines) of CD41 and CD81 cells in blood. C, Anti-Env antibody (Ab)
titers, determined by ELISA, and virus neutralizing (VN) Ab titers, calculated as yield reduction values. D, Frequencies of Gag- (v) and p26.A5(V) specific cytotoxic T lymphocyte precursors (CTLps), determined by stimulation with SIV Gag and normalized to number of CTLp/106 PBMC;
error bars indicate 95% confidence intervals.
JID 1999;180 (October)
Kinetics of SIV-specific CTL
Table 3. Virus detection in blood, lymph nodes, and spleen 22 months
after infection.
a
Virus detection
Monkey
VC
VL
PCR
PBMC
LNMC
SPMC
1
1
1
5
63
25
1
1
1
PBMC
LNMC
SPMC
2
1
1
2
25
13125
1
1
1
PBMC
LNMC
SPMC
2
2
2
2
2
2
2
1
1
PBMC
LNMC
SPMC
2
1
1
2
PBMC
LNMC
SPMC
2
2
1
2
2
K66
K77
K80
1
1
2
1
1
1
2
1
1
K71
a
Virus culture (VC) was done with peripheral blood, lymph node, and spleen
mononuclear cells (PBMC, LNMC, SPMC), and their CD8-depleted fractions
virus load (VL) was determined in serial cell dilutions, and polymerase chain
reaction (PCR) was done with DNA from both freshly isolated and cultured
mononuclear cells and their CD4-enriched fractions.
A remarkable finding was that the frequencies of both SIV
protein- (figures 1 and 2) and epitope- (table 2) specific CTLps
decreased significantly over time in the 3 animals that lost both
culturable virus and PCR-detectable provirus in their PBMC
(i.e., K71, K77, and K80; table 1). These findings suggest that
SIV-specific memory CTLs decline under conditions of limited
antigenic stimulation and bear direct implications for vaccination strategies aimed at inducing protective CTL responses
against lentiviruses. The basis of CTL immunological memory
and the requirement for antigen persistence, however, are a
matter of controversy [19–21]. In fact, evidence has been provided that memory CD81 CTLs may be maintained in the
absence of specific antigen [19]. Nevertheless, our observations
are consistent with the finding that CTL responses against multiple HIV antigens disappear under conditions of limited antigenic expression, as did those found in a long-term nonprogressor carrying a defective HIV provirus [8]. Recently, loss of
tetramer-positive anti-HIV CD81 T cells has also been reported
in HIV-infected patients receiving highly active antiretroviral
therapy [22].
In previous studies, CTLs against a dominant T-cell epitope
have been induced by lower antigenic doses and have lasted
for a longer time than CTLs against minor epitopes [17]. We
found that CTLps against an immunodominant epitope in p26
designated p26.A5 [14] developed more rapidly and lasted
longer in circulation than those directed against other putative
minor Gag epitopes (table 2). These CTLs may require lower
levels of antigenic stimulation [17], be triggered by infected cells
displaying very low epitope density [23], be relatively independent of T cell help [24], and ultimately be more effective in
virus containment [25]. These observations suggest that qualitative aspects of CTL responses are important determinants
of the outcome of the infection. The detection of Rev-specific
CTLps in the 3 nonprogressing macaques but not in the other
animals (table 1) lends further support to this concept. Because
a similar correlation has been observed in HIV-1–infected persons [3], we hope that our preliminary data will encourage
further investigation into the contribution of Rev-specific CTLs
to protection against lentiviruses.
The CTLp decline observed in macaques that effectively contained the infection prompted a study of the outcome of rechallenge. We chose to reinfect only 1 of the 3 nonprogressing
macaques (i.e., K71), so that the other 2 (i.e., K77 and K80)
could provide information on virus distribution in lymphoid
tissues at the end of the study (table 3). When monkey K71
was reinfected with a high dose of homologous virus, transient
viremia with rising CTLp frequencies and antibody titers suggested at least partial susceptibility to reinfection (figure 3).
Although the possibility of virus reactivation cannot be excluded, the temporal association of viremia, decreasing CD41
cells, and increasing CD81 cells, CTLp and antibody titers with
rechallenge makes this possibility less likely. CTLs can be regarded as a very sensitive marker of virus replication. This is
indicated by the finding that CTLps against multiple SIV antigens [26], including the nonvirion proteins Rev and Tat
(E.G.J.H., unpublished data), can be detected in macaques
lacking both detectable virus and antibody responses after
challenge.
After reinfection, virus replication in peripheral blood was
limited in comparison with the virus burst observed during
primary viremia (figure 3). Resistance to superinfection is a
well-described phenomenon in macaques, as reviewed elsewhere
[27]. Although its mechanisms remain unclear, they are likely
to include an immunological component, as reviewed elsewhere
[16]. It is tempting to speculate that restimulated CTLps, rather
then being a mere reflection of the levels of virus replication,
played a role in virus containment after reinfection.
Other mechanisms may be proposed for the observed CTLp
decline in the 3 animals that lost detectable virus in peripheral
blood. Although progressive immunological dysfunction may
result in CTLp decline, the 3 animals showed no signs of disease
progression and maintained stable CD41 cell counts throughout
the study (table 1). Their T cell function, as defined by the
ability to respond to allogeneic cells in mixed-lymphocyte reactions, was preserved over time (not shown). The increase in
CTLp frequencies observed in monkey K71 after reinfection
(figure 3) also argues against a compromised immune function.
During primary infection, HIV-specific CTLs accumulate
preferentially in peripheral blood rather than in lymph nodes
[28]. Consistent with this finding, CTLp frequencies of monkey
K71 were lower in lymph nodes than in blood after both pri-
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Cell type
K88
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Geretti et al.
JID 1999;180 (October)
mary infection and reinfection, whereas splenic CTLp frequencies after reinfection resembled those found in blood (figure 4).
As a consequence of virus containment in the periphery, however, CTLs may home to potential sites of ongoing virus replication, such as lymph nodes [29] and spleen. We studied virus
distribution in these tissues after 22 months of infection (table
3). Although virus was undetectable in peripheral blood from
the 3 nonprogressing macaques, virus reservoirs were found in
their lymphoid organs. This is an important observation, because it confirms that loss of virus detection in peripheral blood
cannot be regarded as an indicator of virus clearance. In the
3 monkeys, however, virus burden in lymphoid tissues was below or barely above the detection limit. In fact, in monkey K71,
it was not dissimilar from that detected in lymph nodes after
primary infection (figure 4). Although CTLp sequestration
seems unlikely in the presence of such a low virus burden, we
aim at extending our study to other macaques and CTLp specificities other than Gag.
In conclusion, our findings add to our understanding of the
dynamics of antiviral CTL responses, suggest a role for SIVspecific CTLs in virus containment, and support the concept
that the quality of antiviral CTLs is an important determinant
of the course of infection.
Acknowledgments
We thank M. P. Cranage and E. W. Rud for providing the SIVmac32H-J5, B. Niesters for continuous support, and C. Kruyssen for
help in preparing the manuscript.
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Kinetics of SIV-specific CTL