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Virology 337 (2005) 222 – 234
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Phenotypic and kinetic analysis of effective simian–human
immunodeficiency virus-specific T cell responses in
DNA- and fowlpox virus-vaccinated macaques
Ivan Stratov, C. Jane Dale, Stephen J. Kent*
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia
Received 21 October 2004; returned to author for revision 14 April 2005; accepted 22 April 2005
Available online 23 May 2005
Abstract
Although T cell immunity is important in the control of HIV-1 infection, the characteristics of effective HIV-specific T cell responses are
unclear. We previously observed protection from virulent SHIV challenges in macaques administered priming with DNA vaccines and
boosting with recombinant fowlpox viruses expressing shared SIV Gag antigens. We therefore performed a detailed kinetic and phenotypic
study of the T cell immunity induced by these vaccines prior to and following SHIV challenge utilizing intracellular cytokine staining. Pigtail
macaques vaccinated intramuscularly with DNA/recombinant fowlpox virus exhibited a coordinated induction of first Gag-specific CD4 T
cell responses and then a week later Gag-specific CD8 T cell responses following the fowlpox virus boost. Overall, the magnitude and timing
of the peak CD8 T cell responses following challenge was significantly associated with reductions in SHIV viremia following pathogenic
challenge. After pathogenic lentiviral challenge, virus-specific effector memory T cells derived from animals controlling SHIV infection
recognized a broad array of epitopes, expressed multiple effector cytokines and rapidly recognized virus-exposed cells ex vivo. These results
shed light on some of the requirements for T cells in the control of pathogenic lentiviral infections.
D 2005 Elsevier Inc. All rights reserved.
Keywords: HIV; Vaccine; Phenotype; T cell; Kinetics; Cytokine ICS; Memory
Introduction
The development of an effective vaccine against HIV-1 is
one of humanity’s greatest challenges. Generating effective
immunity to HIV is hampered by the virus’s capacity to
evade innate, humoral and cell-mediated immunity, the
likely need to generate effective mucosal immunity and the
lack of a precise immune correlate of protection (Nabel,
2001; Stratov et al., 2004).
Although induction of broad neutralizing antibody to
HIV by vaccines would be ideal, this has not been
achievable to date. Many current HIV-1 vaccine research
efforts endeavor to primarily induce effective HIV-specific
T cell immunity. CD8+ cytotoxic T lymphocytes (CTLs)
are important in controlling HIV-1 infection of humans and
* Corresponding author. Fax: +61 383443846.
E-mail address: [email protected] (S.J. Kent).
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.04.023
SIV/SHIV infection in macaques and generally correlate
with protective immunity in macaque-SHIV studies (Barouch et al., 2000; Borrow et al., 1994; Koup et al., 1994;
Ogg et al., 1998). There are several strategies to induce
high-level T cell immunity, including priming with DNA
with or without boosting with recombinant viral/bacterial
vectors or cytokines. Promising data have been obtained in
non-human primate models, showing protection from AIDS
and 100-fold reductions in SHIV viral load following the
induction of SHIV-specific T cell immunity with DNA/
poxvirus prime/boost vaccination regimens (Amara et al.,
2001; Dale et al., 2004a). DNA prime/poxvirus boost
vaccine regimens have provided less durable protection
against SIV in macaques (Horton et al., 2002).
HIV-specific T cell immunity involves both CD4 and CD8
T cells. The absence of functional HIV-specific T cells in
HIV-infected humans is recognized as a key defect in
enabling more efficient control of viremia (Rosenberg et
I. Stratov et al. / Virology 337 (2005) 222 – 234
al., 1997). The safe induction of both CD4 and CD8 T cells is
a critical goal of HIV vaccine studies. HIV-specific CD4 T
cells are likely to enhance the durability, memory levels and
generation of HIV-specific CD8 T cells (Grakoui et al., 2003;
Kalams et al., 1999). The temporal generation of vaccinespecific CD4 T cells would ideally precede and assist the
generation of CD8 T cell responses, but this has not been
previously analyzed in HIV vaccine studies of outbred
macaques.
The significant limitations of relying only on HIVspecific CD8 T cells in controlling HIV has become clear
in recent years. In vivo selection for mutant HIV or SIV viral
variants, which escape CD8 T cell responses, is the rule.
Although T cell escape mutants incur a fitness cost
(Friedrich et al., 2004; Leslie et al., 2004), progression to
AIDS is likely to eventually ensue (Barouch et al., 2002). A
narrowly directed CD8 T cell response is likely to be more
susceptible to T cell escape. The lack of virus-specific CD4
T cells may enhance the generation of T cell escape in HCVprimate models (Grakoui et al., 2003).
The functional phenotype of the CTL response and its
association with outcome is of great interest (McMichael
and Rowland-Jones, 2001). The effector/memory phenotype
of the CTL response is likely to determine both the rapidity
of the CTL response upon infectious virus challenge and the
durability of T cell memory in the setting of long latency
between vaccination and virus exposure. Effector memory
CD8 + T cells (i.e., cells terminally differentiated for direct
anti-pathogen effector activity) have been defined by panels
of surface molecules and are phenotypically CD62L, CCR7
and CD28 negative, CD95 positive and express varying
levels of CD45RA (Pitcher et al., 2002). The cytokine
secretion profile of cognate CTL responses is also a
critically important functional characteristic of effective T
cells (Pantaleo and Koup, 2004). Interferon-gamma (IFNg)
production by CTLs is closely linked to virus-specific
immunity but IFNg production during HIV-1 infection may
still occur even though these cells are defective in their
ability to proliferate or lyse target cells (Appay et al., 2000).
Production of a broader array of cytokines, such as TNFa,
IL-2 and perforin, may be an important determinant of more
effective CD4 T-helper and CD8 CTL responses (McMichael and Rowland-Jones, 2001).
Although most in vitro studies of HIV vaccines analyze
T cell responses to peptide epitopes or peptide pools, for
protective immunity to be optimally effective, T cell
responses should rapidly recognize processed intact virions,
preferably at mucosal surfaces. Phenotyping T cell
responses to processed virions by intracellular cytokine
staining (ICS), which is a short (generally 7 h) assay,
provides an opportunity to study rapid processing and
presentation of the virus itself.
To further understand protective immunity to HIV, we
first evaluated the kinetics of Gag-specific CD4 and CD8 T
cell responses, both before and after SHIV challenge, in
pigtail macaques vaccinated with standard IM regimens of
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DNA and rFPV (Dale et al., 2004a). We subsequently
analyzed the relationship between the virologic outcome of
acute SHIV challenge with the magnitude and timing of the
Gag-specific CD8 T cell response after challenge. To
characterize the SIV/HIV-specific T cell immunity in
successfully immunized macaques further we then analyzed
the T cell immunity in individual animals for their surface
marker phenotype, the number of epitopes recognized, the
cytokine secretion profile and their ability to recognize
intact virions of the T cell responses.
Results
Vaccination and SHIV challenge studies
We studied the kinetics and phenotype of SIV/HIV in
pigtail macaques immunized with DNA prime/FPV boost
vaccine regimens involved in three separate studies (Dale et
al., 2004a; De Rose et al., 2005; Kent et al., submitted). Two
of these studies involved SHIV challenge, the first examining 30 animals receiving combinations of IM DNA and
rFPV vaccinations and a highly pathogenic intrarectal
CXCR4-utilizing SHIV mn229 challenge (Dale et al.,
2004a). The second study involving immunization of 20
macaques with combinations of IM and mucosally delivered
DNA and rFPV vaccines followed by a moderately
pathogenic CCR5-utilizing SHIVSF162P3 challenge. Animals
involved in the 3rd study were immunized IM with various
doses of HIV-1 DNA and rFPV vaccines and were studied
for the cytokine-secretion profile of T cells described below
(De Rose et al., 2005). A summary of the regimens and
outcomes is shown in Table 1.
Based on our previous studies (Dale et al., 2004b; Kent et
al., 1998), we anticipated the most successful vaccination
regimens would be those that used IM priming with two or
three DNA vaccinations followed by one or more rFPV
boosts. This prediction proved true (Table 1). In trial 1, the six
animals vaccinated IM utilizing two DNA priming and with
non-cytokine expressing rFPV boosting (group 1) induced
peak SIVgag-specific CD8+ T lymphocytes capable of
expressing IFNg ranging from 0.3% to 4.7% of all CD8+ T
cells, early after the rFPV boost, as previously reported (Dale
et al., 2004a). After DNA vaccine priming alone, CD8 and
CD4 T cell responses were low (<0.3%) or undetectable.
Kinetics of Gag-specific T cell responses in blood following
prime/boost vaccination
We first studied the kinetic relationship of the SIVgagspecific CD4 and CD8 T cell responses in detail early after the
rFPV boost vaccine in fresh peripheral blood samples from
the six animals in group 1 of trial 1 vaccinated with our most
effective, DNA/rFPV prime/boost regimen. Five of the six
vaccinated animals demonstrated a remarkably coordinated
induction of Gag-specific T cell immune responses (see Fig.
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I. Stratov et al. / Virology 337 (2005) 222 – 234
Table 1
Summary of three separate macaque DNA/fowlpox virus vaccine trials
Trial
Group
Vaccination
regimena
Route of vaccine
(DNA/FPV)
1
1
2 DNA/FPV
IM/IMd
6
2
1 DNA/FPV
IM/IM
6
3
3 DNA
IM/IM
6
4
2 DNA/FPV-IFNg
IM/IM
6
5
Controls
N/A
6
1
3DNA/2FPV
IM/IM
4
2
3DNA/2FPV
IM/IN
4
3
3DNA/2FPV
IM/IR
4
4
3DNA/2FPV
IN/IN
4
5
Controls
N/A
8
3 DNA/FPV
IM/IM
2
3
a
b
c
d
e
n
12e
Animal ID numbers
17105, 4247, 4277,
4290, 4295, 4296
4236, 4237, 4262,
4379, 4380, 4382
4241, 4297, 4299,
4301, 4386, 4523
4243, 4251, 4663,
4664, 4667, 4668
1253, 1281, 4194,
4265, 4529, H3
4245, 4246, 4254,
4381
4240, 4292, 4648,
4666
3790, 4293, 4532,
4635
4238, 4253, 4258,
4259
4250, 4658, 9E, 18E,
19E, 21E, 23E, 24E
200B, 1D52, 710D,
1302, 6820, 4C05,
2976, 3560, 0C18,
5D2F, 505D, 207F
Peak Gag-specific
CD8/CD4 T cell
responsesb
2.4/0.97
Challenge virus
Reduction
in acute
SHIV VLc
Reference
SHIVmn229
IR
1.48
Dale et al.
(2004a)
0.04/0.48
0.84
0.08/0.02
1.53
0.07/0.4
0.88
0/0
N/A
2.33/0.53
SHIVSF162P3
IVag
0.64
0.06/0.11
0.88
0.02/0.04
0.86
0.1/0.07
0.26
0/0
N/A
0.34/1.17
Nil
N/A
Kent et al.
(submitted for
publication)
De Rose
et al.
(2005)
Refers to number of DNA primes and FPV boost vaccines. All vaccines were administered at 3 – 4 weekly intervals.
Mean % T cell response in group.
Mean reduction in peak SHIV plasma RNA (log10 copies/ml plasma) in group compared to control group.
IM = intramuscular; IR = intrarectal; IN = intranasal; IVag = intravaginal.
All animals received the same HIV-1 subtype AE DNA and rFPV vaccines at varying doses.
1A) with an early CD4 T cell response 1 week after the rFPV
boost (mean 1.0%; range 0.2 –2.1%) followed by a larger
(week 2) CD8 T cell response (mean 2.4%; range 0.9– 4.7%).
To confirm this kinetic relationship of the virus-specific
CD4 and CD8 T cell response, we again studied the temporal
association of SIV Gag-specific T cell responses in the four
animals vaccinated IM with the same DNA and rFPV vaccines
in trial 2. The pattern of an initial Gag-specific CD4 T cell
response followed by a CD8 T cell response was repeated
(Fig. 1B). In trial 2, a second boost with rFPV resulted in only
a modest increase in SIVgag-specific CD8 T cells, which did
not reach the level obtained from the initial boosting, likely
due to immunity to the rFPV vector (Dale et al., 2004b).
Kinetics of Gag-specific T cell responses following SHIV
challenge
We then studied the kinetics of recall Gag-specific T cell
responses following SHIV challenge. Following pathogenic
challenge with CXCR4-utilising SHIVmn229, the six animals
in group 1 of trial 1 vaccinated IM twice with DNA and
boosted with rFPV preserved peripheral CD4 T cells and
had lower peak and set point plasma SIV viral loads than
control animals (Dale et al., 2004a). A large anamnestic SIV
Gag-specific CD8 T cell response (mean of 11.7%) was
present in these animals. This response peaked 3 weeks after
challenge and 1 week after the peak of the plasma SHIV
RNA (mean 6.67 log10 copies/mL; Fig. 1C). We also
studied the kinetics of T cell immunity in the animals
vaccinated IM with DNA and rFPV in trial 1 following the
SHIVsf162p3 challenge. A similar pattern was seen in the
four animals in group 1 of trial 2 challenged with CCR5utilising SHIVSF162P3. Peak plasma SIV viral load was 5.50
log10 copies/mL at week 3, closely followed by peak Gagspecific CD8 T cell responses of 7.5% at week 3.5 (data not
shown). Taken together, these kinetic data are consistent
with previous data on the role of CD8 T cells in control of
acute infection (Borrow et al., 1994; Koup et al., 1994).
Post-challenge SIV Gag-specific CD4 T cell responses were
much lower than the CD8 T cell responses and there was no
temporal relationship to the peak viral load or SHIV-specific
CD8 T cell responses (Fig. 1C).
Relationship of CD8 T cell immunity after challenge with
virologic outcome
The temporal relationship between the Gag-specific CD8
T cell response and peak viral load in group 1 animals in
I. Stratov et al. / Virology 337 (2005) 222 – 234
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Fig. 1. Kinetics of mean SIV Gag-specific CD4 (closed circles) and CD8 T cell responses in macaque blood samples (closed diamonds). (A) Following rFPV
boosting, an early (week 1) Gag-specific mean (TSE) CD4 T cell response is detected, followed by a larger and later (week 2) mean Gag-specific CD8 T cell
response detected in the six monkeys in group 1 from trial 1 vaccinated with the standard IM DNA and rFPV regimen. (B) The same temporal pattern of mean
SIV Gag-specific CD4 and CD8 T cell immunity is repeated in the four animals from group 1 in trial 2 vaccinated the same IM DNA and rFPV vaccines as for
trial 1. Note: animals in trial 2 were given a second rFPV boost 4 weeks after the initial rFPV boost. (C) Following SHIVmn229 challenge in trial 1, mean SIV
Gag-specific CD8 T cell responses peak 1 week after peak SHIV plasma viral load (open squares) in the six animals from group 1 vaccinated with the standard
IM DNA and rFPV regimen.
both SHIV challenge trials 1 and 2 suggested that there may
be a correlation between either the timing or strength of T
cell immunity and outcome of challenge. We therefore
analyzed the timing and magnitude of the peak CD8 T cell
response in comparison to the peak level of acute SHIV
viremia. To broaden and improve power for these analyses
we studied all 50 animals (including controls) challenged
with either SHIVmn229 or SHIVSF162P3 across both trials 1
and 2.
The strength of the peak peripheral blood CD8 T cell
post-challenge was indeed significantly inversely correlated
with the peak SHIV viral load across the 50 animals infected
in both trials 1 and 2 with the SHIV challenge viruses (Fig.
2A). We then also assessed whether the timing of the postchallenge peak CD8 immunity affected the peak level of
SHIV viremia post-challenge. Animals with a very early
(week 1) or late (4 weeks) peak peripheral blood CD8 T
cell response following SHIV challenge were less likely to
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I. Stratov et al. / Virology 337 (2005) 222 – 234
Fig. 2. Post-challenge correlation between peak SHIV plasma viral load and gag-specific CD8 T cell IFNg responses in PBMC. All 50 control and
vaccinated macaques receiving the various DNA and rFPV regimens in both trial 1 and trial 2 (see Table 1) were included in these analyses. (A) The peak
viral load is inversely correlated with the peak IFNg response (P = 0.003; R-squared 0.18). (B) The peak viral load correlates with the timing of the peak
IFNg response (P = 0.014, t test).
control the peak SHIV viremia, with a mean 0.88 log10
copies/mL higher level of plasma SHIV RNA compared to
animals with peak SIV Gag-specific CD8 T cell responses
that peaked at weeks 2 – 3 (Fig. 2B).
Phenotype of Gag-specific T cells
To further characterize effective T cell immunity to
SIV/HIV in macaques, we then performed additional
experiments on the phenotype, breadth, cytokine secretion
profile and ability of T cell responses to recognition of
whole virions. For these analyses, we studied some of the
animals vaccinated with the DNA/FPV vaccine regimens
(group 1 of trials 1 and 2) associated with high levels of
peripheral T cell immunity and good outcomes from SHIV
challenge.
To first determine the phenotype of the effector CD8 T
cells to SIV Gag assisting in the control of SHIV viremia,
we studied the surface expression of a series of molecules
associated with an effector/memory phenotype in an ICS
assay on PBMC from animals in group 1 of trial 1. The
h-chain of CD8 is specific to CD3+8+ T lymphocytes.
CD8hhi T cells are more characteristic of naı̈ve cells, with
increased expression in cord blood and larger Vh repertoire
or they express markers of central memory (CCR7, CD62L
and CD28), while CD8hlo T cells display an effector
memory phenotype with increased levels of activation and
cytotoxic molecules and express low levels of CD62L and
CCR7 but high levels of surface CD95 (Konno et al., 2002;
Schmitz et al., 1998). The vast majority of the responding
CD8 T cells were confined to the CD8hlo-expressing cells
(Fig. 3A). These effector SIVgag-specific CD8 T cells were
phenotypically CD45RA negative and CD95 positive
(Fig. 3B). To study CD62L expression, a marker of effector
T cells (Kaech et al., 2002), PBMC were sorted for CD62L
expression (high or low) prior to ICS assay to obviate
CD62L down regulation during the 7 h in vitro incubation
for the ICS assay. CD8 T cells producing IFNg in response
I. Stratov et al. / Virology 337 (2005) 222 – 234
227
Differential cytokine secretion of Gag-specific T cells
IFNg is only one of several effector cytokines likely to be
important in the control of lentivirus infections, with
evidence that TNFa and IL-2 are potentially important
mediators of effective control of these infections (McKay et
al., 2002; Pantaleo and Koup, 2004). We therefore studied
the differential cytokine profile of Gag-specific T cells in
DNA/rFPV-vaccinated macaques (Fig. 4).
In 9 of 11 blood samples studied in animals in group 1 of
trials 1 and 2, DNA/rFPV vaccine-induced Gag-specific
CD4 and CD8 T cells simultaneously produced both IFNg
and TNFa (Fig. 4A). We then also studied the kinetics of the
simultaneous production of IL-2, IFNg and TNFa after the
Fig. 3. Phenotype of SIV Gag-specific T cell responses in peripheral blood
by intracellular cytokine staining. Whole blood was stimulated with
overlapping Gag peptides and lymphocytes gated by forward and sidescatter criteria and their phenotype analyzed by four-color flow cytometry
using combinations of CD3-PE, CD8h-PE, CD45RA-PE, CD62L-PE,
CD8a-PerCP, CD95-FITC and IFNg-APC. For all analyses, macaques from
group 1 of trial 1 were studied, with representative plots of one animal from
at least four animals studied shown for panels A and B and two animals for
panel C. (A) Five weeks following pathogenic SHIV challenge, a large Gagspecific CD3+CD8a+ IFNg immune response (left plot, using CD3-PE,
CD8a-PerCP, CD4-FITC and IFNg-APC) is induced in animals previously
vaccinated with DNA followed by rFPV boosting. When the Gag-specific
cells (gated directly from lymphocytes, using CD8h-PE, CD4-FITC and
IFNg-APC) were analyzed for CD8h expression (right panel), the majority
of the Gag-specific cells expressing IFNg were contained within the CD8hlo
population. (B) Two weeks post-rFPV boost, CD8a+lymphocytes were
studied for CD45RA and CD95 expression—Gag-specific lymphocytes are
CD45RA negative (right plot, using CD45RA-PE, CD8a-PerCP, CD4-FITC
and IFNg-APC) and CD95 positive (left plot, using CD95-FITC, CD3-PE,
CD8a-PerCP and IFNg-APC). (C) Twenty-nine weeks after SHIV
challenge, cells were initially sorted for CD62L expression prior to ICS
assay and then CD8a expressing T lymphocytes studied for Gag-specific
IFNg expression (using CD3-PE, CD8a-PerCP, CD4-FITC and IFNgAPC)—Effector cells are CD62Llo (right plot).
to SIV Gag following SHIV challenge were confined to the
sorted cells that were CD62L negative (0.34% Gag-specific
CD8 T cells cf. 0.05% for CD62Lhi; Fig. 3C).
Fig. 4. Differential cytokine production by SIV/HIV-specific T cells. (A)
Representative flow cytometric data from animal 4295 (group 1, trial 1) 18
weeks post-SHIV challenge: CD8 Gag-specific peripheral blood T cells
expressing IFNg also produced TNFa. (B) Time course of HIV Gagspecific T cell responses post-rFPV boosting (and prior to challenge) of a
representative macaque in trial 3; IL-2 production from peripheral blood T
cells paralleled the expression of IFNg and TNFa in both CD8- and CD4positive T cells.
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I. Stratov et al. / Virology 337 (2005) 222 – 234
rFPV in two animals vaccinated with HIV-1 expressing
DNA and rFPV vaccines in trial 3. IL-2 was also produced
by Gag-specific T cells although we did not co-stain for all
three cytokines in the same tube. Nevertheless, the
production of all three cytokines by Gag-specific CD4 and
CD8 T cells followed the same temporal pattern after rFPV
boosting when studied kinetically in macaques vaccinated
with HIV-1 DNA and rFPV vaccines in trial 3, although the
production of IL-2 was shorter-lived (Fig. 4B). A similar
pattern was observed for both animals studied and this was
duplicated even when individual epitopes were tested, as
opposed to a pool of peptides (data not shown).
There were, however, interesting exceptions to this
general observation that IFNg secretion from Gag-specific
T cells paralleled that of other cytokines. In 2 of 11
animals studied, Gag-specific CD8 T cells primarily
liberated TNFa and not IFNg (Fig. 5). After the rFPV
boosting, DNA-primed animals 4245 and 4381 (from trial
2, group 1, respectively) demonstrated minimal or no
(0.02% and 0.03%, respectively) CD8 + T cells expressing
IFNg in response to SIV Gag but did demonstrate TNFa
responses of 0.27% and 0.42%, respectively. In these two
animals, the Gag-specific CD4 T cell responses secreted
both IFNg and TNFa. Even after SHIV challenge,
macaque 4245, which controlled SHIVSF162P3 plasma
viremia to <3.2log10 copies/mL by 11 weeks after
challenge, failed to mount Gag-specific CD8+ T cells
expressing IFNg, although Gag-specific CD4 T cells
expressing IFNg were again detected. Although anecdotal,
this observation suggests that secretion of TNFa by SHIVspecific T cells may, at least in some animals, facilitate
control of viremia.
Breadth of SHIV-specific CD4 and CD8 T cell responses
Macaques receiving DNA/rFPV vaccination (primarily
expressing shared SIV Gag/Pol antigens) are able to develop
effective CD8 and CD4 T cell responses following SHIV
challenge virus (Dale et al., 2004a). Responses were
detected against multiple SIV/HIV proteins in many
animals, including peptide pools derived from HIV-1 Env,
SIV Pol and Nef as well as SIV Gag, although responses
directed against Gag were the strongest, presumably
reflecting better priming of Gag-specific T cells by the
Gag-expressing vaccines (data not shown).
Sufficient data were generated in many animals to study,
in detail, the breadth of epitopes recognized within the Gag
protein. Utilizing truncated sets of overlapping 15mer
peptides, at least 19 different CD8 and 18 different CD4
epitopes were detected (Fernandez et al., 2005). An example
of epitope mapping in a macaque with a broad and robust
Gag-specific T cell response 4277 (group 1, trial 1) is shown
in Fig. 6. This macaque recognized a minimum of seven
epitopes (three CD8 and four CD4 epitopes) on initial
screening with pools of five peptides. Of the seven epitopes
identified (including the peptide 41 epitope which had been
detected in other macaques), six of these T cell responses
were narrowed to single 15mer peptides or to a pair of
overlapping 15mer peptides. We have recently restricted one
of the peptide epitopes (peptide 41) to a novel pigtail
macaque MHC class I molecule ManeA*10 (Smith et al.,
2005) and work on MHC restriction of other epitopes is
ongoing.
Overall, the breadth of the T cell response across IM
vaccinated macaques in group 1 of trials 1 and 2 averaged
Fig. 5. Primary production of TNFa and not IFNg by SIV Gag-specific CD8 T cells in 2 of 11 macaques studied. Gated CD3/CD8a peripheral blood
lymphocytes, obtained 3 weeks after the rFPV boost, were analyzed for Gag-specific cytokine expression by ICS. The percentage of non-CD8a T cell
responses noted in the upper left quadrants reflect CD4+ T cells. Both macaques were from trial 2, group 1.
I. Stratov et al. / Virology 337 (2005) 222 – 234
Fig. 6. Broad SIV Gag-specific T cell responses in PBMC following DNA/
rFPV vaccination and SHIV challenge. At week 14 following SHIV
challenge, animal 4277 (group 1, trial 1) was studied for T cell immunity to
pools of five 15mer SIV Gag peptides, overlapping by 11 amino acids. The
percentage of CD8 or CD4 T cells expressing IFNg in response to peptide
pools or individual peptides are shown. Many of these T cell responses were
mapped to single 15mer peptides or two overlapping 15mer peptides in
subsequent weeks (arrows); confirmed positive results are in bold. Specific
amino acid sequences of the epitopes were as follows: Peptide 1/2 overlap
NSVLSGKKADE; Peptide 4/5 overlap EKIRLRPNGKK; Peptide 14/15
overlap CQKILSVLAPL; Peptide 48/49 overlap VGDHQAAMQII; Peptide 78 QTDAAVKNWMTQLL. A number of likely additional CD4
epitopes (underlined) were also present but responses not narrowed to
single or adjacent 15mers.
3.3 Gag epitopes per animal in peripheral blood. In contrast,
only 2 of the 10 controls across both trials 1 and 2 had a
sufficient Gag-specific T cell response after challenge to
map T cell epitopes, and in both of these animals
recognition of only a single 15mer peptide each was
detected.
Recognition of whole SIV by CD8 and CD4 T cells
We then studied the Gag-specific CD4 and CD8 T cell
immune responses induced by DNA/rFPV vaccination for
their ability to recognize SIV/HIV in its native conformation, rather than just as individual 15mer peptides. Using
conformationally intact whole inactivated SIV, we investigated the time required in vitro for T cells in whole blood
to respond to processed whole SIV virions. The time taken
to process and present SIV was evaluated by adding BFA,
which blocks intracellular processing of whole antigen for
presentation by MHC class I to CD8 T cells, at varying time
intervals after exposure of antigen. A macaque, 4290 from
229
group 1 in trial 1, which had a favorable outcome from
SHIV challenge with persistent high levels of both CD8 and
CD4 Gag-specific T cell immunity was studied for these
experiments.
Within 30 min antigen-presenting cells in peripheral
blood processed and began to present epitopes of the
whole virus on their surface for recognition by circulating
CD8 T cells (Fig. 7A). CD8 T cell expression of
intracellular IFNg in response to whole inactivated SIV
peaked with a 2-h processing time prior to the addition of
BFA. The CD8 T cell responses to the SIV Gag peptide
pool were 10-fold greater than that to whole SIV,
presumably reflecting the higher molarity of peptide
epitopes available. Unlike responses to whole SIV, CD8
T cell responses to the Gag peptide pool are comparatively
unaffected by addition of BFA at time zero. This suggests
that the responses to the whole inactivated SIV particle are
not due to contaminating free Gag peptide fragments. The
CD4 T cell responses to the whole inactivated SIV
parallels, and is nearly equivalent to, the CD4 T cell
response to free Gag peptides, indicating that alternative
processing of class II antigens from the whole inactivated
SIV for CD4 recognition does not require such extensive
intracellular processing (Fig. 7B).
Discussion
Protection from disease, at least in the short term, is
observed following pathogenic SHIV challenge of macaques using prime/boost vaccine regimens (Dale et al.,
2004a; Robinson et al., 1999; Shiver et al., 2002).
Several previous studies have either measured SHIVspecific T cell immune responses by IFNg ELISpot,
which is unable to readily phenotype effector cells, or
focus primarily on measuring the magnitude of the CD8
T cell response to a single immunodominant peptide by
MHC class I tetramer analysis (Barouch et al., 2000).
Vaccines based on single viral epitopes have been
relatively inefficient in protecting macaques against SIV
challenge (Yasutomi et al., 1995) and there is increasing
evidence for CTL escape from narrowly directed CD8 T
cell immunity (O’Connor et al., 2002). Further defining
the phenotype and kinetics of protective CD4 and CD8 T
cell immune responses may assist rational improvement
of vaccine design.
The systematic study of SIV Gag-specific CD4 and CD8
T cells by ICS following rFPV boost in standard IM
regimens of DNA and rFPV vaccinations in 10 macaques
across two trials revealed an interesting pattern. We
observed a coordinated induction of first SIV Gag-specific
CD4 T cell immune responses, followed by larger peaks of
CD8 T cell immune responses. Whether this relationship
between CD4 and CD8 T cell immune responses also occurs
with other viral proteins remains to be determined and likely
to depend in part on the efficiency of vaccination, epitopes
230
I. Stratov et al. / Virology 337 (2005) 222 – 234
Fig. 7. Short interval of processing of whole inactivated SIV for presentation to Gag-specific T cells. A time course of IFNg production by T cells in PBMC
obtained from macaque 4290 (trial 1, group 1) 37 weeks post-challenge to either SIV Gag 15mer peptide pool or whole inactivated SIV. (A) CD8+ responses to
whole inactivated SIV increase over time; peak responses occur with a 2- to 4-h time delay prior to the addition of BFA. (Note log scale). (B) CD4 T cell
responses to free peptide and whole inactivated SIV parallel each other, reflecting different cellular processing.
encoded by each protein and MHC haplotype of the host.
However, such a temporal relationship should be desirable
in maintaining memory levels of CD8 T cell responses,
given evidence that CD4 T cell help is critical in the
expansion of CD8 + CTLs upon re-encounter with antigen
(Janssen et al., 2003). Janssen et al. demonstrated that mice
depleted of CD4 T cells prior to secondary encounter with
antigen can still mount CD8 + CTL responses provided
CD4 help was available during the initial encounter with
antigen during immunization. This is of critical importance
given that acute HIV infection is tropic for CD4 T cells
rendering them dysfunctional (Douek et al., 2002).
The kinetics and magnitude of the anamnestic Gagspecific T cell response following SHIV challenge was also
intriguing. For these studies we examined the relationship of
peak CD8 T cell responses after SHIV challenge with peak
acute SHIV viremia levels in 50 control and vaccinated
animals across two trials. The timing (weeks 2 –3) and
magnitude of the peak Gag-specific CD8 T cell response
following challenge were significantly associated with
improved control of SHIV viremia across the two SHIV
challenge studies performed. Optimal generation and
expansion of virus-specific CD8 T cell responses following
encounter with pathogenic lentiviruses is likely to be an
important determinant of the outcome of infection. Interestingly, there was no clear relationship between the recall of
SHIV-specific CD4 T cell responses and control of peak
viremia, possibly because these cells are readily infected,
and likely rendered dysfunctional, by primate lentiviruses
(Douek et al., 2002).
The phenotype and cytokine of Gag-specific CD8 T cells
following challenge was further probed by studying
individual macaques vaccinated with DNA and rFPV
regimens associated with successful outcomes of SHIV
challenge. As expected, the phenotype was that of an
effector/memory T cell, having the surface cell phenotype of
CD8hlo, CD45RA negative, CD95hi and CD62L negative.
The proportion of SIVgag-specific cells in the CD8hlo
fraction was approximately twofold higher than that in the
CD3/CD8a fraction. Additional surface markers such as
CCR7 would have been useful to confirm this phenotype
but were not available for pigtail macaque cells, although
CD8hlo cells are typically CCR7 negative (Konno et al.,
2002). Generally, all three cytokines studied (IFNg, TNFa
I. Stratov et al. / Virology 337 (2005) 222 – 234
and IL-2) were expressed by Gag-specific T cells, both after
vaccination and after challenge. Expression of IL-2 was
more short lived in both CD4 and CD8 T cells after the
rFPV boost compared to IFNg and TNFa expression,
consistent with their effector/memory phenotype (Konno et
al., 2002). IL-2 expression is likely to drive expansion of the
virus-specific T cells and may wane as vaccine-expressed
antigen levels decline. More prominent IL-2 expression
from virus-specific T cells is a feature of central memory
cells (Konno et al., 2002; Sallusto et al., 1999) and may
result in more durable levels of T cell immunity.
There were exceptions observed to the general rule that
multiple cytokines are co-expressed by Gag-specific T cells.
Two of 11 animals studied predominantly expressed TNFa
and not IFNg, and one of these animals achieved full control
of viral replication. Although requiring confirmation in
larger studies, this observation suggests that TNFa expression may be sufficient for effective immunity and perhaps
an improved surrogate marker for effective T cell immunity.
However, we consistently noted mildly higher levels of
background TNFa expression from T cells in unstimulated
blood (data not shown), compared to IFNg expression,
which limits the sensitivity of TNFa expression in studying
antigen-specific cells.
A large breadth of SIV-specific CD4 and CD8 T cell
responses was also observed following SHIV challenge of
IM DNA/rFPV-vaccinated macaques. A broad T cell
response is likely to be more important than a large
narrowly focussed one. Rapid recognition of whole virions
by both CD4 and CD8 T cell responses prior to release
of progeny viruses should be critical in effectively controlling viremia (Yang et al., 1996, 1997). We studied T
cell recognition of whole processed virions. SIV-specific
CD8 T cells induced by DNA/rFPV vaccination begin to
secrete IFNg within 30 min, indicating rapid processing of
whole inactivated SIV virions within antigen-presenting
cells. SIV-specific CD4 T cells do not require processing
of SIV through the endoplasmic reticulum and can express
IFNg when BFA is added simultaneously with the
antigen. This rapid recognition of virus exposed cells by
SIV-specific CD8 and CD4 T cells is likely to be an
important factor in the control of virus replication. Future
studies could address the comparative rapidity of recognition of virus-infected cells by specific T cell responses
to more accurately define the requirements for protective
immunity.
In summary, this study of DNA/rFPV-vaccinated macaques found a coordinated induction of broad CD4 and CD8
T cell responses. Recall of Gag-specific CD8 T cell immune
responses correlated with reductions in SHIV viremia
following pathogenic challenge. The broad T cell responses
induced were of an effector-memory phenotype, capable of
expressing multiple effector cytokines and rapidly recognized virus-exposed cells. Taken together, these results
illustrate some of the potentially complex requirements of
effective T cell immunity to HIV-1.
231
Materials and methods
Animal experimentation
Studies were performed on juvenile pigtail macaques
(Macaca nemestrina). All experimentation was approved by
relevant ethics committees under previously described
conditions (Dale et al., 2002, 2004a).
Vaccine protocols
Three separate pigtail macaque vaccine trials studying
DNA/rFPV prime/boost vaccination were undertaken using
various protocols as previously described (Dale et al.,
2004a; Kent et al., 1998) (see Table 1). Briefly, trial 1
involved two intramuscular DNA vaccine priming and
boosting with a single intramuscular rFPV vaccine (both
encoding shared SIVmac239 Gag/Pol proteins), followed by
rectal challenge with highly pathogenic SHIVmn229 which
results in reductions in SHIV viremia and retention of CD4
T cells (Dale et al., 2002, 2004a; Kent et al., 2002). Trial 2
involved three DNA priming vaccinations then two boosting
vaccinations with rFPV (again encoding shared SIV
proteins), followed by vaginal challenge with moderately
pathogenic SHIVSF162P3 (Harouse et al., 1999, 2001), which
also resulted in reduction in SHIV viral load in comparison
to control unvaccinated animals (S. Kent et al., manuscript
submitted). Trial 3 involved three intramuscular DNA
priming vaccinations and a single intramuscular rFPV
boosting, both encoding HIV-1 subtype AE proteins, without viral challenge (De Rose et al., 2005).
DNA vaccines
DNA vaccine, pHIS-SHIV-B, encoded full-length unmutated SIVmac239 Gag and Pol, HIV-1NL43, Tat, Rev and Vpu
and the 5V one third of HIV-1AD8 Env (Dale et al., 2004a).
Genes were inserted into vector pHIS-64 (Dr. Heather
Davis, Coley Pharmaceuticals) behind the human cytomegalovirus immediate –early promoter. Plasmid vector pHIS64 has kanamycin resistance, bovine growth hormone
polyA termination signal and 14 primate-optimized CpG
immunostimulatory sequences. Empty vector plasmid DNA,
pHIS, served as a control vaccine. Plasmid pHIS-SHIV-B
(1mg/dose IM) for immunization was prepared by QIAGEN
(Hilden, Germany), control DNA vaccine pHIS was
prepared using the EndoFree Plasmid Giga kit (QIAGEN).
Recombinant fowlpox virus vaccines
Construction of the FPV vaccines has been described
(Boyle et al., 2004; Dale et al., 2004a). Briefly, FPVgag/pol,
expressing SIV Gag/Pol, was constructed by inserting the
promoter-SIV gag/pol PCR amplicon into pKG10aSIVgagpol for insertion into FPV-M3 at the F6,7,9 site. PCR
primers used the FPV early/late promoter and an early
232
I. Stratov et al. / Virology 337 (2005) 222 – 234
transcription terminator for insertion into fowlpox virus
vector FPV-M3. HIV-193TH254 env (mutated to remove the
middle third) was amplified from plasmid vector
pCH34AEenv(m) and inserted into a separate FPV-M3
vector at the REV site. Recombinants were selected on the
basis of co-expression of the Escherichia coli gpt gene and
plaque purified on the basis of co-expression of the E. coli
beta-galactosidase gene. FPV vaccines were prepared in
saline.
Antigens
Antigens studied for T cell responses were primarily
15mer peptides overlapping by 11 amino acids spanning
SIVmac239 Gag, Pol and Nef, HIV-1MN Env, all kindly
obtained from the NIH AIDS Research and Reference
Reagent Repository. For trial 3 we obtained homologous
subtype AE HIV-19TH253 Gag 15mer peptides (Auspep,
Parkville, Australia). Peptides were solubilized in pure
DMSO at high concentrations (1 mg peptide/10 –40 AL
DMSO) and peptides for each antigen pooled. 1 Ag/mL of
each 15mer peptide within the pools was used for the ICS
assay. To assess immune responses to intact virions we used
whole aldrithiol-2 inactivated SIVmne (Arthur et al., 1998;
Dale et al., 2004a) (10 Ag/mL, kindly provided by Dr J
Lifson, National Cancer Institute, Frederick, MD) and the
relevant microvesicle control preparation derived from the
same cell line used to grow the virus.
Intracellular cytokine staining
Intracellular cytokine staining (ICS) assay was performed as previously described (Dale et al., 2004a; Maecker
et al., 2001). Briefly, in the presence of co-stimulatory
molecules CD28 and CD49d (BD Sciences), whole blood or
mononuclear cell preparations were incubated with pools of
15mer peptides or whole inactivated SIV. Brefeldin A
(BFA; Sigma; final concentration 10 Ag/mL) was added to
prevent protein export from the endoplasmic reticulum.
Incubation times were generally terminated after 7 h, the
last 5 h in the presence of BFA, although this varied in some
experiments. Cells were then stained with surface antibodies
including anti-CD4 FITC and PerCP, anti-CD3 PE, antiCD8 PerCP and APC, anti-CD45RA PE, anti-CD95 FITC,
anti-CD62L PE (BD Biosciences, Pharmingen, San Diego,
CA) or CD8h PE (Immunotech, Marseille, France), then
lysed and permeabilized as per standard protocols. Intracellular staining was then performed with anti-IFNg APC,
anti-IL-2 APC and/or anti-TNF-a FITC (BD) and fixed
prior to acquisition via FACSort (BD) and analyzed using
cell quest software (BD).
Determining SHIV-specific T cells by ICS
Gating on T lymphocytes was performed using forward
and side scatter criteria and combined this with surface
expression of CD3, based on the nadir of a CD3 histogram.
Two-dimensional dot plots (x-axis: CD8; y-axis: IFNg)
were then formed and responses calculated using quadrant
lines. The vertical quadrant line (defining CD3/CD8+ cells)
was placed along the margin where the density of clearly
CD8+ cells started to reduce, so as to be confident that all
cells included in the quadrant were truly CD8+, although
very similar percentages were obtained when the vertical
quadrant line was placed at the nadir of a CD8+ cell
histogram (not shown). The horizontal quadrant line
(defining IFNg-producing cells) was placed within approximately 10 channel values above the margin where the
density of CD8+ cells had clearly reduced. Analyses
generally were performed after collecting approximately
100,000 lymphocytes. This produced a final CD3/CD8+
count of approximately 20,000 events; counts of less than
1000 CD3/CD8 (or CD3/CD4) events were excluded from
analysis.
Validation of true positive antigen-specific responses
Validation of the ICS assay in M. nemestrina was
performed by studying background levels of IFNg
production in the first 101 negative control samples; one
(1%) sample was excluded from our statistical validation
of the cut-off for a true positive. This sample had a
background of 1.88% (i.e., 48-fold above the mean
background level) and was 5.7-fold larger than the next
largest background level of 0.33%. From these 100
samples used for final statistical analysis, 81% of negative
control samples had a background 0.05%, the mean
background being 0.04% (range 0.00 –0.33%; standard
deviation 0.05%). Ninety-six (96%) of the samples had a
background level within 2 standard deviations of the mean.
We determined that a true positive was an antigen-specific
result more than two standard deviations above the
background level (i.e., approximately 0.10% or threefold
above background). To be confident of a true positive in
samples with backgrounds greater than 0.05%, we determined that IFNg production in antigen-specific samples
needed to be >0.10% above background and threefold
above background.
Background ICS results were consistent throughout the
studies in naı̈ve, vaccinated and SHIV-infected macaques;
that is, 80 –90% of samples had background levels <0.05%
and only 1 – 2% of samples had very high backgrounds
(>10-fold above average background).
Cell sorting to determine CD62L status
Sorting for cells for the expression of CD62 ligand
(CD62L) was performed on a MoFlo flow cytometer (Dako
cytomation, Fort Collins, USA). Briefly, fresh PBMC were
stained with CD62L-PE (at 4 -C for 30 min) and CD62Lpositive and -negative cells sorted into separate tubes before
proceeding to Gag antigen stimulation.
I. Stratov et al. / Virology 337 (2005) 222 – 234
Acknowledgments
We thank Rob De Rose, Socheata Chea, Caroline
Fernandez, Richard Sydenham, Alistair Ramsay, Charani
Ranasinghe, Scott Thomson, Ian Ramshaw, Barbara Coupar, David Boyle, Matthew Law and all other members of
the Australian Thai HIV Vaccine Consortium for helpful
assistance and advice. Supported by the NIH, a division of
the US Department of Health and Human Services, award
N01-AI-05395 and Australian National Health and Medical
Research Council grants 299907, 251653 and 251654.
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