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k Hel*
* Basic Research Laboratory, National Cancer Institute, Bethesda, MD 20892;
Third Department of Pediatrics, Medical Academy of Bialystok, Bialystok, Waszyngtona, Poland;
Southern Research Institute, Frederick, MD 21701;
Center for AIDS Research, Duke University Medical Center, Durham, NC 27710;
¶ Biostatistics and Data Management Section, National Cancer Institute, Bethesda, MD 20892;
|| Aventis-Pasteur, Toronto, Ontario, Canada;
# Weill Medical College, Cornell University, New York, NY 10021; and
** Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5 mo) was treated with continuous antiretroviral therapy (ART). A
group of eight macaques was vaccinated with or without simultaneous
administration of low dose IL-2 with the highly attenuated poxvirus
vector (NYVAC) vaccine candidate expressing the SIVmac structural
gag-pol-env (gpe) genes and a novel
chimeric fusion protein derived from the rev-tat-nef
(rtn) regulatory genes. Control groups consisted of
mock-vaccinated macaques or animals treated only with IL-2. Vaccination
significantly expanded both virus-specific CD4+ and
CD8+ T cell responses, and IL-2 further increased the
vaccine-induced response to an immunodominant Gag epitope. Following
antiretroviral treatment interruption, the viral set point was
significantly lower in vaccinated than in control macaques for at least
4 consecutive mo, and viral containment was inversely correlated with
vaccine-induced, virus-specific CD4+ and CD8+ T
cell responses. These data provide the proof of concept that
therapeutic vaccination before cessation of ART may be a feasible
approach in the clinical management of HIV-1
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To this end we have chosen the highly attenuated NYVAC-based
recombinant vaccines SIV-gag-pol-env (SIV-gpe)
and SIV-rev-tat-nef (SIV-rtn;
NYVAC-SIV-gpe (19) and NYVAC-SIV-rtn
(20)), because of the ability of this vaccine modality to
induce/expand virus-specific CD4+ and
CD8+ T cell responses in naive macaques
(19, 21) as well as in SIVmac251-infected macaques
(17, 52). During primary SIVmac251 infection, the possible
benefit of immunization with this vaccine modality is obscured by the
beneficial effect of early initiation of ART, as also observed in
some HIV-1-infected individuals treated early in infection
(17, 22, 23, 24, 25, 26, 27, 53, 54). Therefore, we enrolled macaques
infected with SIVmac251 for 6 mo, treated them with ART, and
immunized them without or with simultaneous administration of IL-2 or
with mock NYVAC vaccine. In humans, administration of daily low dose
(i.e., 
2 million U/day) of IL-2 has been found to augment
cell-mediated immune responses and to increase the number of
circulating NK cells as well as CD4+ T cells
(28). In addition, when administered before and after
structured treatment interruption (STI), daily low dose IL-2 resulted
in increased circulating CD8+ T cell
concentrations coincident with a decline in plasma HIV concentrations
to a set point (29). In parallel, other macaques infected
with the same viral stocks (17, 30) that had been viremic
for a minimum of 5 mo to a maximum of 15 mo were treated either with
ART and IL-2 or with ART alone. Antiviral therapy cessation was used as
a diagnostic tool to assess the immune control on viral replication in
all macaque groups.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ART consisted of i.v. administration of didanosine (10
mg/kg/day), oral administration of stavudine twice a day (1.2
mg/kg/dose), and s.c. administration of
(R)-9-(2-phosphonylmethoxypropyl) adenine (20 mg/kg/day).
NYVAC-SIV-gpe, NYVAC-SIV-rtn (20),
or NYVAC vector was given by the i.m. route at a dose of 1 x
108 PFU/immunization to macaques that were not
subjected to prior treatment except for infection with SIVmac251(561)
(30) and ART treatment, as indicated in Fig. 1
A. Macaques in groups 3 and 4
had been infected with the same stock of SIVmac251(561)
(30) for a minimum of 5 mo to a maximum of 15 mo, and
their previous treatment was described previously (17, 30). IL-2 was given daily by s.c. inoculation at a dose of
120,000 IU to macaques in groups 3 and 4. The dose was decided
following a pharmacokinetic study in macaques (9, 55).
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Eepitope-specific CD3+
CD8+ T lymphocytes were detected by tetramer
staining. Fresh PBMC were stained with anti-human CD3 Ab (PerCP
labeled, clone SP34; BD PharMingen, San Diego, CA), anti-human
CD8
Ab (FITC labeled; BD Bioscience, San Jose, CA), and Mamu-A*01
tetrameric complexes refolded in the presence of a specific peptide and
conjugated to PE-labeled streptavidin (Molecular Probes, Eugene, OR).
Gag181–189 CM9 (CTPYDINQM) (Gag CM9) and
Tat28–35 SL8 (TTPESANL; Tat SL8)-specific
tetramers were used (18). Samples were analyzed on a
FACSCalibur (BD Biosciences), and the data are presented as the
percentage of tetramer-positive cells of all CD3+
CD8+ lymphocytes.
ELISPOT assay
The Mamu-A*01-restricted peptides
Gag181–189 CM9, Gag QI9, Env TL9, and Tat SL8
(31) were used in Mamu-A*01-positive macaques. In
Mamu-A*01-negative macaques, peptide pools (15 aa in size overlapped by
11 aa) encompassing the entire Gag, Env, Tat, and Rev proteins were
used. One to 2 µg of each pool were used (32). The pools
contained 120 peptides for Gag, 100 for Env 1, 96 for Env 2, 22 for
Tat, and 20 for Rev. Information on the precise composition of each
pool is available upon request. Macaque IFN-
-specific ELISPOT kits
(U-Cytech, Utrecht, The Netherlands) were used to detect the number of
Ag-specific, IFN--producing cells. Ninety-six-well, flat-bottom
plates were coated with anti-IFN- mAb MD-1 overnight at 4°C
and blocked with 2% BSA in PBS for 1–3 h at 37°C. Cells (1 x
105/well) were loaded in triplicate in RPMI 1640
containing 5% human serum and specific peptides or 5 µg/ml Con A as
a positive control. The plates were incubated overnight at 37°C in
5% CO2 and developed according to the
manufacturer’s guidelines (U-Cytech).
Intracellular cytokine staining
PBMC (1 x 106) were incubated
for 2 h at 37°C with 1 µg/ml of peptides in RPMI 1640 growth
medium containing 5% FBS, 0.5% gentamicin, and 5% glutamine.
Unstimulated or staphylococcal enterotoxin B-stimulated PBMC served as
negative and positive controls, respectively. To allow for
intracellular accumulation of IFN-, 2 µl Golgiplug
(brefeldin A; BD PharMingen) was added, and the incubation was
continued overnight at 37°C. PBMC were then stained for
CD4+ and CD8+ T cell
subsets using anti-human CD4 and CD8 mAbs conjugated to FITC
(CD4-FITC, catalog no. 71024L; BD PharMingen) and PerCP (CD8-PerCP,
catalog no. 347314; BD Biosciences), respectively. After 30-min
incubation at 4°C, PBMC were washed in PBS, fixed, and permeabilized
with 100 µl Cytofix/Perm solution (BD PharMingen), followed by
staining for intracellular IFN- using 0.4 µg of PE-conjugated
anti-human IFN- (BD PharMingen). Finally, PBMC were washed,
resuspended in PBS containing 2% formaldehyde, and analyzed using a
FACSCalibur flow cytometer and CellQuest software. By generating dot
plots of forward vs side scatter, 30,000 events were acquired in the
lymphocyte population from which IFN--positive
CD4+ and CD8+ T lymphocytes
were calculated. Results were expressed as the percentage of
IFN--positive cells for each lymphocyte
subset.
Neutralizing Ab assay
Neutralizing Abs against the primary SIVmac251/561L (produced in human PBMC) were detected as described previously (19). Neutralizing Ab titers were defined as the reciprocal plasma dilution at which 50% of the target cells were protected from virus-induced killing as detected by neutral red uptake. CEMx174 cells were used as targets for SIVmac251 virus, and CEMxR5 were used as targets for SIVmac251/561L virus.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nineteen macaques infected with the SIVmac251 (561)
challenge stock (30) were enrolled in the study and
divided into five groups (Fig. 1
A). The macaques in groups
1, 2, and 3 were naive before infection with SIVmac251. Macaques in
groups 4 and 5 were infected with the same viral strain as
previously described (17, 30). The macaques were subjected
to a daily triple drug regimen of the nucleotide analogs
(R)-9-(2-phosphonylmethoxypropyl) adenine, stavudine (d4T),
and didanosine for a minimum of 21 wk (Fig. 1). IL-2 at low dose was
chosen to promote immune restoration (33, 34) and
potentiate the vaccine effect. One group of macaques was immunized with
the NYVAC mock vaccine (group 1), and two groups were immunized with
the NYVAC-SIV-gpe and NYVAC-SIV-rtn vaccines
without (group 2) or with (group 3) coadministration of daily low dose
IL-2 (120,000 IU; Fig. 1A). Macaques in group 4 received
IL-2 only, and those in group 5 had no additional treatment except ART
(Fig. 1B). In macaques from groups 4 and 5, a short
interruption of ART was performed between wk 21 and 28 before
suspending all treatments, as indicated in Fig. 1B.
At the start of ART treatment neither the absolute
CD4+ T cell count per cubic millimeter nor the
plasma viral RNA copy number differed significantly (by Kruskal-Wallis
test, P > 0.2) among macaques enrolled in the groups
to be vaccinated (groups 2 and 3) or controls (group 1; Fig. 1, C and D, and Table I), and plasma virus levels became
undetectable (<2000 copies/100 µl) during ART in all three groups of
macaques. Viral rebound in macaques in groups 4 and 5 occurred when ART
was suspended between wk 21 and 28 (data not shown).
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Ten of the 19 macaques had the MHC class I Mamu-A*01 molecule
(35, 36) (Fig. 1A), thereby enabling us to
measure Mamu-A*01-restricted CD8+ T cell
responses to dominant and subdominant SIVmac epitopes (31)
using tetramers (36, 37), ELISPOT, or intracellular
cytokine staining. In the nine Mamu-A*01-negative macaques, the
virus-specific CD8+ and
CD4+ T cell responses were assessed by ELISPOT
and intracellular cytokine staining (38) following in
vitro stimulation with 15 mers overlapping 11 aa (39)
encompassing the Gag, Env, Rev, Tat, and Nef proteins of SIVmac251.
Proliferative responses to p27 Gag and gp120 Env were also measured in
all 19 macaques.
ART treatment decreased the percentage of
Gag181–189 CM9 tetramer-positive cells in blood,
which, at the time of the first vaccination, was at the background
level in Mamu-A*01-positive macaques from groups 1, 2, and 3 (Fig. 1E). The first immunization with NYVAC-SIV-gpe
(wk 42) significantly increased the percentage of
CD8+ T cells specific for the
Gag181–189 CM9 epitope in the blood of macaques
from groups 2 and 3. Following the second immunization (wk 48), a
significant additive effect of IL-2 coadministration was observed in
macaques from group 3 (p = 0.016; wk 49–54;
Fig. 1E). However, at the time of STI (10) (wk
58), the mean frequency of this response in the blood of the immunized
macaques was equivalent in macaques from groups 2 and 3 (Fig. 1E). The Tat SL8 response was also expanded by vaccination
with NYVAC-SIV-rtn (Fig. 1F); however, in this
case, no significant differences were observed among macaques treated
or not with IL-2. No significant changes in the frequency of the
responses to Gag181–189 CM9 or Tat SL8 were
observed during ART treatment of macaques in groups 4 and 5 (data not
shown).
At the start of immunization (wk 42), the cumulative frequency of
virus-specific T cells with various antigenic specificities measured by
ELISPOT varied among macaques and were increased significantly in the
vaccinated macaques. Repeated measures ANOVA of the square
root-transformed ELISPOT values from weekly measurements (wk 42–58)
that included various specificities (Gag, Env, Tat, Rev) demonstrated
significant differences among the mock-vaccinated and vaccinated
macaques (p = 0.023). ELISPOT data for various
specificities for each individual macaque at the time of immunization
(wk 42) and at the maximum response are shown in Fig. 2A. Macaques vaccinated in the
presence of IL-2 did not develop significantly higher ELISPOT responses
measured with Gag, Env, Tat, and Rev overlapping peptides than those
vaccinated in the absence of the cytokine over the entire period of
immunization (wk 42–58; Fig. 2A and data not shown).
Similarly, no significant differences in the kinetics or duration of
ELISPOT response were observed when the vaccine was given together with
IL-2 (data not shown).
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was also assessed by direct intracellular IFN-
staining following short term in vitro stimulation with peptide pools
encompassing the entire SIVmac Gag and Env proteins. At the time of
immunization (wk 42), both CD8+ and
CD8- (CD4+) cumulative
Gag- and Env-specific responses were <0.5% in all macaques except
macaque 3143, which had mounted naturally the highest and broadest
CD8+ T cell responses by wk 42 (Fig. 2B). Both CD8+ and
CD8- (CD4+) T cell
responses to Gag and Env pools were significantly increased at wk 44
above the baseline in the vaccinated groups (p
= 0.0096 for CD4+ T cell responses and
p = 0.0005 for the CD8+ T cell
responses). In the mock-vaccinated macaques neither
CD4+ nor CD8+ T cell
responses significantly increased at 2 wk after the first mock
immunization. Similarly, measurements performed at wk 53, 5 wk after
the second immunization (Fig. 2B), confirmed that vaccinated
animals had increased Gag- and Env-specific CD8+
T cell responses (p = 0.0021). Interestingly, a
marginal improvement in the ability of CD8+ T
cells to produce IFN- was also observed in the mock-vaccinated
macaques (p = 0.032), suggesting that ART
treatment may improve this virus-specific functional response.
Because in both macaques and humans treated with ART some
reconstitution of the virus-specific proliferative response to Gag has
been observed (15, 17, 26), we assessed whether IL-2
and/or vaccination could further expand this response (Fig. 2C). Following immunizations, proliferative responses to
both Gag and Env were significantly increased in the vaccinated
macaques from group 2 compared with those in mock-vaccinated macaques
in group 1 (p27 Gag, p < 0.0001; gp120 Env,
p = 0.0002; by repeated measures ANOVA)
during the interval of wk 43–57. Surprisingly, however, in macaques
vaccinated in the presence of IL-2, virus-specific proliferative
responses were significantly lower than those in macaques from group 2
(p27 Gag, p = 0.0039; gp120 Env, p =
0.015; by repeated measures ANOVA) within the same interval. In fact,
no significant difference in the Gag proliferative response was
observed in mock-vaccinated (group 1) and vaccinated
macaques (group 3) treated with IL-2, and only a marginally significant
difference was observed in the gp120 proliferative response
(p = 0.039) between these two groups. Low
proliferative responses were measured in macaques in groups 4 and 5
(data not shown).
These findings raised the possibility that in IL-2-treated macaques,
Gag- or Env-specific proliferative responses might not necessarily go
hand in hand with the ability of Gag- or Env-specific
CD4+ Th cells to produce IFN-. In fact, for
example, although the frequency of Gag- and Env-specific
CD4+ T cells producing IFN- was increased by
vaccination in IL-2-treated macaque 3069 (see data at wk 44 in Fig. 2B), at the same time points the proliferative response to
Gag was quite low (stimulation index = 5.5 for p27 Gag and at
background level for Env). A scatter plot of lymphoproliferative
responses for Gag and Env vs IFN- production by
CD8- (CD4+) T cells
suggested a direct correlation between these responses (Fig. 2, D and E). The Spearman rank correlation
coefficient was 0.60 (p = 0.21) for p27 and
0.67 (p = 0.15) for gp120, but this direct
correlation did not reach statistical significance, in part because of
the very small number of animals tested. This apparent discordance
needs to be further confirmed in studies with a larger number of
macaques. Discordance between the proliferative response and the
ability of virus-specific CD4+ Th cells to
produce cytokine has also been observed in HIV-1-infected individuals
(38).
Prior vaccination is associated with improved viremia containment following ART suspension
ART was suspended in the macaques at the time indicated in Fig. 1, A and B. Plasma virus rebounded in all macaques,
and the overall extent of plasma virus during the first 2 wk of STI did
not differ significantly in macaques from all groups (Fig. 3, A and B).
However, the viral set point (wk 4–11) was significantly lower
(p = 0.024, by Wilcoxon rank-sum test) in the
vaccinated macaques (groups 2 and 3) than in control macaques (Fig. 3, A and C) and remained significant up to wk 16
(p = 0.042). The comparisons of set point
viremia among groups 1, 2, and 5 and between groups 3 and 5 did not
reach statistical significance because of the small numbers of
macaques. However, when these two comparisons were combined in a
stratified test of the vaccine’s effect, with IL-2 as the
stratification factor, the difference was again significant at the
p = 0.027 level. A clear additive effect of IL-2 with
vaccination, although suggested by the low viremia in these macaques,
could not be properly evaluated because one of the three macaques (3056
in group 2) died due to drug-related toxicity at 3 wk after STI, and
the number of animals became insufficient for that statistical
evaluation of the data.
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, A and B), indicating that treatment with IL-2
and ART alone did not provide a virological benefit. To further
increase the strength of the statistical analysis, virus levels after
STI in all 11 control macaques (groups 1, 4, and 5) were compared with
those in the eight vaccinated macaques (groups 2 and 3). A
significantly lower viremia in the vaccinated macaques was again
observed (p = 0.026), further confirming that
vaccination before STI, but not ART or IL-2 treatment alone, is
associated with a decrease in viral set point within the first weeks of
STI. While these data provide the first proof that therapeutic
immunization may ameliorate the virological outcome, the effect of
vaccination was not durable, and by 9 mo after STI the virus plasma
levels were equivalent in control and vaccinated macaques (data not
shown). Immune correlates of viremia containment during STI
As demonstrated above, viremia was contained significantly better in vaccinated macaques within the first 4 mo of STI. Since the significance of median log viremia over eight time points (wk 4–11) was 0.024, and the p value between wk 8–15 varied between 0.012 and 0.042, we chose the median virus load at wk 4–11 as representative of a set point value from the middle of the range to perform correlative analysis with immune response preceding and following STI.
The analysis of ELISPOT data measured weekly in all macaques
after immunization (wk 42–58) included responses specific for the
entire Gag, Env, Tat, and Rev proteins in Mamu-A*01-negative macaques.
In the case of the Mamu-A*01-positive macaques, the analysis included
ELISPOT responses to the Gag181–189 CM9, QI9,
Env TL9, and Tat SL8 epitopes (31). A significant inverse
correlation (p = 0.04) was found between the
median of all measured SIV-specific CD4+ and
CD8+ T cell responses (wk 42–58) measured by
ELISPOT induced by vaccination and plasma virus levels at the set point
during ART suspension (Fig. 4A).
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Proliferative responses to p27 Gag or gp120 elicited by vaccination
were highest following the first immunization and were not boosted
significantly by the second and third immunizations (Fig. 2C). Analysis of peak proliferation values at wk 44 for each
macaque and median virus load at set point during STI revealed negative
Spearman rank correlation coefficients of -0.61 and -0.59 for p27 Gag
and gp120, respectively. However, although the coefficients were
noteworthy in size, the p values were only close to the
standard threshold for statistical significance
(p = 0.048 and p = 0.057,
respectively; Fig. 4, B and C).
Next we measured neutralizing Ab titers against the primary viral
challenge, SIVmac251(561) (30) before ART initiation and
during viral rebound and assessed whether they correlated positively or
negatively with viremia containment at the set point. Serum
neutralizing Ab titers to the primary viral stock challenge before ART
initiation and at 1–2, 3, 9, or 11–13 wk after therapy suspension are
summarized in Table II. Overall, viral
rebound elicited the highest titers of neutralizing Abs in the
vaccinated macaques of group 1. Analysis of the correlation between set
point viremia and neutralizing Ab titers at 4 times after ART
suspension and set point resulted in negative correlation coefficients
of -0.32 (p = 0.34), -0.52
(p = 0.098), -0.19 (p
= 0.58), and -0.33 (p = 0.32) from the
earliest to the latest points measured (Table II). However, a positive
trend was apparent between the pre-ART titers and set point viremia
(correlation coefficient = 0.53; p = 0.068). Thus,
Ab titers to the primary SIVmac251(561) (30) challenge
stock that pre-existed and/or were expanded within the first 4 wk of
STI may have contributed to viremia containment during STI. A similar
association was described in a few HIV-1-infected individuals
(18).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, both virus-specific CD4+ and
CD8+ T cell responses inversely correlated with
viremia containment even in a study with a relatively low number of
subjects, as also observed in a prophylactic vaccine study in
macaques in which NYVAC-SIV-gpe was used in combination with
DNA (21, 56). The frequency of Gag-specific
CD8+ immune T cells, whether measured with a
specific tetramer or by a functional readout such as intracellular
cytokine staining assay (IFN- production), inversely correlated with
viremia containment. Both the Gag-specific proliferative responses and
the intracellular cytokine staining of CD8-
(CD4+) T cells expanded by vaccination correlated
negatively with set point viremia after STI and directly with each
other. As expected, the overall response measured by ELISPOT using
specific peptides recognized by Mamu-A*01-positive macaques
(31) or overlapping tetramers (11 aa overall) encompassing
the entire Gag, Env, Tat, and Rev proteins also inversely correlated
with set point viremia during STI. A negative correlation, although not
significant, was also found between neutralizing Ab titers to the
primary SIVmac251(561) (30) viral challenge stock, which
is as difficult to neutralize as other SIVmac isolates (our unpublished
observations), also suggesting that neutralizing Ab may have
contributed to viremia containment. Thus, possibly all adaptive immune
responses might have contributed to the transient reduction of viremia
observed in the vaccinated macaques.
Importantly, vaccination did not worsen the virological outcome. Since HIV-1-specific CD4+ T cells appear to be preferentially infected by the virus (40), vaccination by inducing a significant expansion of virus-specific CD4+ T cells in the macaques studied here could have resulted in higher viremia after STI. The facts that a reduction of viremia was indeed observed in our study and that it inversely correlated with virus-specific CD4+ T cell responses suggest that the magnitude and functionality of this response induced by vaccination were sufficient to improve, at least transiently, the function of CD8+ T cells, which is known to be impaired in SIVmac as well as HIV-1 infection (41, 42, 43, 44, 45), and might have resulted in a decreased spread of the virus to CD4+ T cells. Indeed, CD4+ T cells are essential in maintaining a functional immune response (46), and in the SIVmac model adaptive transfer of naive CD4+ T cells has provided virological benefit (47).
The transient nature of immune control observed in our study also
suggests that more potent vaccine strategies or a combination of
vaccine modalities is probably needed to obtain protracted virological
benefit. To this end, here we have also attempted to modulate the
response to vaccination using a low dose of IL-2. Collectively, our
results demonstrate that the immune response elicited by this vaccine
modality was modulated by the simultaneous administration of a
continuous low dose of IL-2. The addition of IL-2 to NYVAC-SIV-based
vaccines was associated with a significant increase in the frequency of
tetramer-staining CD3+ CD8+
T cells to the dominant Gag181–189 CM9 response
in the blood of macaques at the peak of the immune response following
the second immunization. However, at the end of the immunization
regimen, no difference in the frequency of CD8+
memory cells with this specificity was observed in macaques vaccinated
in the presence of IL-2 (Fig. 1E). A higher number of
IFN--producing cells in the ELISPOT following the
Gag181–189 CM9-specific peptide stimulation was
also observed during the same period, suggesting that IL-2 modulated
the expansion/survival of this vaccine-induced functional response to a
dominant Gag epitope.
Analysis of the frequency of ELISPOT responses with other
specificities, such as to the entire Gag, Env, Tat, and Rev proteins,
in the vaccinated macaques, however, did not demonstrate significant
differences in the extent or duration of this response in macaques also
treated with IL-2. Similarly, measurement of intracellular production
of IFN- in CD4+ or CD8+
T cells following in vitro stimulation with a peptide pool encompassing
the entire Gag and Env proteins did not reveal differences in macaques
vaccinated in the presence of low dose IL-2. However, we believe that
these questions need to be addressed in a larger number of macaques
before reaching any definitive conclusion.
Surprisingly, however, significant differences were observed in the
extent of the proliferative response to both p27 Gag and gp120 (Fig. 2C). Although the underlying mechanism of this phenomenon is
at present unclear, it is possible that IL-2 treatment, by increasing
the activation and the turnover of Ag-specific cells in vivo (48, 49), may result in a decreased ability of these cells to further
proliferate in vitro. Alternatively, IL-2 may induce cell death in vivo
(50) and truly decrease the frequency of these
CD4+ Ag-specific T cells. We believe that the
latter interpretation is unlikely, since at the end of the immunization
regimen the proliferative responses in the vaccinated groups were
equivalent (Fig. 2C). Nevertheless, because direct
quantification of CD4+ T cell responses to Gag
and Env, using the intracellular cytokine staining method, in parallel
with lymphoproliferative responses was not performed in a sufficient
number of macaques, further studies are needed to clarify this point
(Fig. 2D).
Even though in this study we were unable to demonstrate by statistical methods that the addition of IL-2 to the vaccine improved the virological outcome after STI (because of the drug-related death of one macaque), macaques treated with the combination of IL-2 and vaccine appeared to contain viremia effectively during STI, suggesting that the addition of IL-2 did not worsen the virological outcome. Thus, the relative efficacy of low dose IL-2 as a vaccine adjuvant needs to be further evaluated in a larger study. To this end, the use of intermittent dosing vs continuous administration of this cytokine as well as other cytokines that may increase the frequency and function of Ag-specific cells in combination with vaccination also needs to be carefully evaluated. In summary, we believe that active vaccination may be of benefit to ART-treated individuals with long-standing HIV-1 infection, and we hope that the data presented here will be instrumental in moving various vaccine modalities, alone or in combination with cytokines, to clinical testing in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Genoveffa Franchini, Section of Animal Models and Retroviral Vaccines, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 41 Library Drive, Building 41, Room D804, Bethesda, MD 20892. E-mail address: franchig{at}mail.nih.gov
3 Abbreviations used in this paper: ART, antiretroviral therapy; gpe, gag-pol-env; rtn, rev-tat-nef; STI, structured treatment interruption.
4 G. Franchini. 2002. Modeling immune intervention strategies for HIV-1 infection of humans in the macaque model. Submitted for publication.
5 M. Markowitz, X. Jin, A. Hurley, V. Simon, B. Ramratnam, M. Louie, G. R. Deschenes, M. Ramanathan, Jr., S. Barsoum, J. VanderHoeven, et al. 2002. Discontinuation of antiretroviral therapy commenced early in the course of HIV-1 infection with or without adjunctive vaccination. Submitted for publication.
Received for publication June 14, 2002. Accepted for publication August 27, 2002.
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  M. Vaccari, J. Mattapallil, K. Song, W.-P. Tsai, A. Hryniewicz, D. Venzon, M. Zanetti, K. A. Reimann, M. Roederer, and G. Franchini Reduced Protection from Simian Immunodeficiency Virus SIVmac251 Infection Afforded by Memory CD8+ T Cells Induced by Vaccination during CD4+ T-Cell Deficiency J. Virol., October 1, 2008; 82(19): 9629 - 9638. [Abstract] [Full Text] [PDF]
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 V. Cecchinato, E. Tryniszewska, Z. M. Ma, M. Vaccari, A. Boasso, W.-P. Tsai, C. Petrovas, D. Fuchs, J.-M. Heraud, D. Venzon, et al. Immune Activation Driven by CTLA-4 Blockade Augments Viral Replication at Mucosal Sites in Simian Immunodeficiency Virus Infection J. Immunol., April 15, 2008; 180(8): 5439 - 5447. [Abstract] [Full Text] [PDF]
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 S.-J. Ha, S. N. Mueller, E. J. Wherry, D. L. Barber, R. D. Aubert, A. H. Sharpe, G. J. Freeman, and R. Ahmed Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection J. Exp. Med., March 17, 2008; 205(3): 543 - 555. [Abstract] [Full Text] [PDF]
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A. Boasso, M. Vaccari, A. Hryniewicz, D. Fuchs, J. Nacsa, V. Cecchinato, J. Andersson, G. Franchini, G. M. Shearer, and C. Chougnet Regulatory T-Cell Markers, Indoleamine 2,3-Dioxygenase, and Virus Levels in Spleen and Gut during Progressive Simian Immunodeficiency Virus Infection J. Virol., November 1, 2007; 81(21): 11593 - 11603. [Abstract] [Full Text] [PDF]
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 C. Petrovas, D. A. Price, J. Mattapallil, D. R. Ambrozak, C. Geldmacher, V. Cecchinato, M. Vaccari, E. Tryniszewska, E. Gostick, M. Roederer, et al. SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection Blood, August 1, 2007; 110(3): 928 - 936. [Abstract] [Full Text] [PDF]
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A. Hryniewicz, D. A. Price, M. Moniuszko, A. Boasso, Y. Edghill-Spano, S. M. West, D. Venzon, M. Vaccari, W.-P. Tsai, E. Tryniszewska, et al. Interleukin-15 but Not Interleukin-7 Abrogates Vaccine-Induced Decrease in Virus Level in Simian Immunodeficiency Virusmac251-Infected Macaques J. Immunol., March 15, 2007; 178(6): 3492 - 3504. [Abstract] [Full Text] [PDF]
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A. S. von Gegerfelt, M. Rosati, C. Alicea, A. Valentin, P. Roth, J. Bear, G. Franchini, P. S. Albert, N. Bischofberger, J. D. Boyer, et al. Long-Lasting Decrease in Viremia in Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 after Therapeutic DNA Immunization J. Virol., February 15, 2007; 81(4): 1972 - 1979. [Abstract] [Full Text] [PDF]
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A. Hryniewicz, A. Boasso, Y. Edghill-Smith, M. Vaccari, D. Fuchs, D. Venzon, J. Nacsa, M. R. Betts, W.-P. Tsai, J.-M. Heraud, et al. CTLA-4 blockade decreases TGF-beta, IDO, and viral RNA expression in tissues of SIVmac251-infected macaques Blood, December 1, 2006; 108(12): 3834 - 3842. [Abstract] [Full Text] [PDF]
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K. K. A. Van Rompay, R. P. Singh, W. Heneine, J. A. Johnson, D. C. Montefiori, N. Bischofberger, and M. L. Marthas Structured treatment interruptions with tenofovir monotherapy for simian immunodeficiency virus-infected newborn macaques. J. Virol., July 1, 2006; 80(13): 6399 - 6410. [Abstract] [Full Text] [PDF]
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L. Dorrell, H. Yang, B. Ondondo, T. Dong, K. di Gleria, A. Suttill, C. Conlon, D. Brown, P. Williams, P. Bowness, et al. Expansion and Diversification of Virus-Specific T Cells following Immunization of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals with a Recombinant Modified Vaccinia Virus Ankara/HIV-1 Gag Vaccine. J. Virol., May 1, 2006; 80(10): 4705 - 4716. [Abstract] [Full Text] [PDF]
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M. Vaccari, C. J. Trindade, D. Venzon, M. Zanetti, and G. Franchini Vaccine-Induced CD8+ Central Memory T Cells in Protection from Simian AIDS J. Immunol., September 15, 2005; 175(6): 3502 - 3507. [Abstract] [Full Text] [PDF]
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D. Mavilio, J. Benjamin, D. Kim, G. Lombardo, M. Daucher, A. Kinter, E. Nies-Kraske, E. Marcenaro, A. Moretta, and A. S. Fauci Identification of NKG2A and NKp80 as specific natural killer cell markers in rhesus and pigtailed monkeys Blood, September 1, 2005; 106(5): 1718 - 1725. [Abstract] [Full Text] [PDF]
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E. J. Wherry, J. N. Blattman, and R. Ahmed Low CD8 T-Cell Proliferative Potential and High Viral Load Limit the Effectiveness of Therapeutic Vaccination J. Virol., July 15, 2005; 79(14): 8960 - 8968. [Abstract] [Full Text] [PDF]
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J. J. Donnelly, B. Wahren, and M. A. Liu DNA Vaccines: Progress and Challenges J. Immunol., July 15, 2005; 175(2): 633 - 639. [Abstract] [Full Text] [PDF]
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J. Nacsa, Y. Edghill-Smith, W.-P. Tsai, D. Venzon, E. Tryniszewska, A. Hryniewicz, M. Moniuszko, A. Kinter, K. A. Smith, and G. Franchini Contrasting Effects of Low-Dose IL-2 on Vaccine-Boosted Simian Immunodeficiency Virus (SIV)-Specific CD4+ and CD8+ T Cells in Macaques Chronically Infected with SIVmac251 J. Immunol., February 15, 2005; 174(4): 1913 - 1921. [Abstract] [Full Text] [PDF]
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D. R. Casimiro, A. J. Bett, T.-m. Fu, M.-E. Davies, A. Tang, K. A. Wilson, M. Chen, R. Long, T. McKelvey, M. Chastain, et al. Heterologous Human Immunodeficiency Virus Type 1 Priming-Boosting Immunization Strategies Involving Replication-Defective Adenovirus and Poxvirus Vaccine Vectors J. Virol., October 15, 2004; 78(20): 11434 - 11438. [Abstract] [Full Text] [PDF]
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E. J. Wherry and R. Ahmed Memory CD8 T-Cell Differentiation during Viral Infection J. Virol., June 1, 2004; 78(11): 5535 - 5545. [Full Text] [PDF]
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 S. Kinloch-de Loes Role of therapeutic vaccines in the control of HIV-1 J. Antimicrob. Chemother., April 1, 2004; 53(4): 562 - 566. [Abstract] [Full Text] [PDF]
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M. Moniuszko, C. Brown, R. Pal, E. Tryniszewska, W.-P. Tsai, V. M. Hirsch, and G. Franchini High Frequency of Virus-Specific CD8+ T Cells in the Central Nervous System of Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 J. Virol., November 15, 2003; 77(22): 12346 - 12351. [Abstract] [Full Text] [PDF]
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 I. M. Belyakov, P. Earl, A. Dzutsev, V. A. Kuznetsov, M. Lemon, L. S. Wyatt, J. T. Snyder, J. D. Ahlers, G. Franchini, B. Moss, et al. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses PNAS, August 5, 2003; 100(16): 9458 - 9463. [Abstract] [Full Text] [PDF]
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S. Oh, J. A. Berzofsky, D. S. Burke, T. A. Waldmann, and L. P. Perera Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity PNAS, March 18, 2003; 100(6): 3392 - 3397. [Abstract] [Full Text] [PDF]
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