Nef-deleted mutants of simian immunodeficiency
virus (SIV) and simian and human immunodeficiency
chimeric virus (SHIV) were found to be effective live-
attenuated vaccine candidates in macaque models (9,
11, 24). We previously reported that a new nef-deleted
SHIV-NI is nonpathogenic and that it totally protected
macaques from challenge infection with a nef-intact
parental NM-3rN and partially protected them from
challenge infection with heterologous pathogenic
SHIV-C2/1 (54, 55). To increase protective vaccine
efficacy by immune-potentiation, a nef-deleted SIV or
SHIV expressing IFN-γ was constructed and tried (13,
23, 49). In addition, the other researcher examined
immunization with co-immunization of plasmid vectors
Protective Efficacy of Nonpathogenic Nef-Deleted
SHIV Vaccination Combined with Recombinant
IFN-? Administration against a Pathogenic SHIV
Challenge in Rhesus Monkeys
Kentaro Kaneyasu1, Masakazu Kita2, Sadayuki Ohkura1, Toshiro Yamamoto2, Kentaro Ibuki1,
Yoshimi Enose1, Akihiko Sato3, Makoto Kodama3, Tomoyuki Miura1, and Masanori Hayami*, 1
1Institute for Virus Research, Kyoto University, Kyoto, Kyoto 606–8507, Japan, 2Department of Microbiology, Kyoto Prefec-
tural University of Medicine, Kyoto, Kyoto 602–8566, Japan, and 3Discovery Research Laboratories, Shionogi & Co., Ltd.,
Osaka, Osaka 566–0022, Japan
Communicated by Dr. Hidechika Okada: Received September 29, 2005. Accepted October 11, 2005
Abstract: We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques
protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-? augments
the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of
recombinant human IFN-? (rIFN-?) in vaccinated and unvaccinated monkeys. Nine monkeys were vacci-
nated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-? and the
other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1,
CD4?T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated
monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were
reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-? compared with those in
SHIV-NI vaccinated monkeys without rIFN-?. The peaks of plasma viral load were inversely correlated
with the number of SIV Gag-specific IFN-?-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-?,
the number of SIV Gag-specific IFN-?-producing cells of PBMCs increased 2-fold compared with those in
SHIV-NI-vaccinated monkeys without rIFN-?, and the NK activity and MIP-1? production of PBMCs
were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-? was more effective in
modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These
results suggest that IFN-? augmented the anti-viral effect by enhancing innate immunity and shifting the
immune response to Th1.
Key words: Adjuvant, Cytokine, SHIV, IFN-?
Microbiol. Immunol., 49(12), 1083–1094, 2005
Abbreviations: AIDS, acquired immunodeficiency syndrome;
ELISPOT, enzyme-linked immunospot; ELISA, enzyme-linked
immunosorbent assay; HIV, human immunodeficiency virus;
MIP-1α, macrophage inflammatory protein-1α; NK, natural
killer; PBMC, peripheral blood mononuclear cell; SFCs, spot-
forming cells; SHIV, simian/human immunodeficiency virus;
SIV, simian immunodeficiency virus; TCID50, 50% tissue cul-
ture infectious dose.
*Address correspondence to Dr. Masanori Hayami, Laboratory
of Primate Model, Experimental Research Center for Infectious
Disease, Institute for Virus Research, Kyoto University, 53
Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Kyoto 606–8507,
Japan. Fax: ?81–75–761–9335. E-mail: mhayami@virus.
expressing IFN-γ with SIV antigens (17, 30).
IFN-γ is a cytokine that possesses antiviral activity,
including activity against HIV in vitro (18, 51). IFN-γ,
in addition to having a direct effect on virus replication,
also affects the course of infection and induction of pro-
tective immunity by modulating the antiviral immune
response including macrophage activating activities and
T cell growth promoting activity (8, 10, 18, 19, 21, 26).
The release of IFN-γ early in an infection was reported
to contribute to differentiation of T cells to Th1 cells
(56). IFN-γ is critical for the induction of cell-mediated
immunity, especially cytotoxic-T cell (CTL) responses
(6, 33, 46), and was reported to be one of the main
effector molecules released by CTLs after antigenic
We have previously reported that an SHIV having
human IFN-γ inserted into the deleted nef region
(SHIV-IFN-γ) had a greater protective effect against a
challenge infection with a heterologous pathogenic
SHIV-C2/1 than nef-deleted SHIV having no IFN-γ
(13). These results raised a possibility that IFN-γ
increases the suppression of heterologous pathogenic
In the present study, to investigate whether IFN-γ
augments the immune response induced by vaccination
with a live attenuated SHIV, we examined the antiviral
effect of rIFN-γ administration and adjuvant effect of
rIFN-γ in live attenuated SHIV-vaccinated or unvacci-
nated monkeys. An enzyme-linked immunospot
(ELISPOT) assay was used to analyze SIV Gag-specific
immune responses elicited in monkeys either by rIFN-γ
administration alone or SHIV-NI vaccination combined
with rIFN-γ administration. Moreover, to investigate
whether IFN-γ affects the innate immunity, we exam-
ined the natural killer (NK) cell activity and production
of CC-chemokines and cytokines.
Our results show that SHIV-NI vaccination com-
bined with rIFN-γ administration is effective for induc-
ing a stronger protective cellular immune response
against SHIV-C2/1, suggesting that it has promise as a
potential vaccine adjuvant. In addition, the enhance-
ment of NK activity and MIP-1α production may act
together as a functional unit enhancing the innate and
adaptive immunity to drive a type 1 immune reaction in
SHIV-NI-vaccinated monkeys with rIFN-γ.
Materials and Methods
Monkeys. Rhesus macaques (Macaca mulatta) about
4 kg in weight, were used in this experiment. All mon-
keys were maintained according to the institutional ani-
mal care and use guidelines of the Institute for Virus
Research, Kyoto University.
Virus preparation. SHIV-C2/1 is an SHIV-89.6 vari-
ant isolated by passage at the peak of initial plasma
viremia from an infected cynomolgus macaque (47).
SHIV-C 2/1 virus stock was prepared from the culture
supernatants of COS-7 cells transfected with an SHIV-
C2/1 molecular clone, pKS661 and was stored in liquid
nitrogen until use. SHIV-NI obtained by deleting the
nef gene from SHIV-NM-3rN, was used as an attenuat-
ed vaccine virus (13, 23). SHIV-NM-3rN is a chimeric
simian and human immunodeficiency virus, having the
envelope gene of HIV-1 pNL432. These virus stocks
were prepared from supernatants of a human T cell line,
M8166, transfected with pSHIV-NI.
Interferon-γ. ImunomaxR-γ, which contains rIFN-γ
3?106U/ml, maltose 52.6 mg/ml and L-cysteine 0.44
mg/ml was a kind gift from Shionogi & Co., Ltd.,
Osaka, Japan. As solutes, maltose 52.6 mg/ml and L-
cysteine 0.44 mg/ml which was used as placebo. Plas-
ma IFN-γ concentrations were determined using a com-
mercially available ELISA kit (BioSource International,
Camarillo, Calif., U.S.A.).
Pharmacokinetics of exogenous IFN-γ in rhesus
macaques. Plasma rIFN-γ levels were assessed sequen-
tially after single intramuscular administration of 1?106
U/ml rIFN-γ to two macaques. Subsequently, plasma
rIFN-γ levels in rhesus macaques were measured with
an immunoassay kit (BioSource International). The
rIFN-γ concentrations in rhesus macaques remained at
high levels (100 pg/ml to 80 pg/ml) until 10 hr after
injection. This level is similar to the level that was
found to be effective against cancer (53).
Immunization and challenge. Four treatments were
given (Table 1). To examine the antiviral effect of
rIFN-γ administration against a pathogenic SHIV-C2/1
infection, three of five unvaccinated monkeys were
intramuscularly administered with rIFN-γ (group A)
dissolved in 5% auto-monkey serum and the other two
(group B) were given placebo dissolved in solvent of
rIFN-γ. rIFN-γ was administered at a dose of 1?106
U/ml per monkey three times per week for 4 weeks. To
investigate whether IFN-γ augments the immune
response induced by immunization with a live attenuat-
ed SHIV, nine monkeys were intravenously inoculated
with 1?105TCID50 of SHIV-NI. Four of them were
given rIFN-γ (group C) and the other five were given
placebo (group D). Subsequently all groups were intra-
venously challenged with 200 TCID50of the pathogenic
SHIV-C2/1. One monkey of group C (MM411) died
by accident after the challenge. This monkey did not
show complete loss of CD4?T cells which is a symp-
tom of AIDS.
Sample collection. Peripheral blood mononuclear
cells (PBMCs) were prepared by Percoll density gradi-
K. KANEYASU ET AL
ent centrifugation. Freshly isolated PBMCs were used
for ELISPOT assay and NK assay as described below.
All plasma samples were frozen at ?80 C until use.
Quantification of plasma viral RNA loads. Plasma
viral RNA loads were determined by quantitative RT-
PCR (50). Total RNAs were prepared from plasma
with a QIAamp viral RNA kit (QIAGEN) according to
the manufacturer’s recommendations, and RT-PCR was
performed using a Taqman RT-PCR kit (Perkin Elmer).
RNAs of attenuated viruses and challenge virus were
evaluated with primer pairs specific to SHIV NM-3rN
and SHIV-C2/1, respectively, as previously described
(14). These reactions were performed with a Prism
7700 Sequence Detector (Applied Biosystems, Foster
City, Calif., U.S.A.) and analyzed using the manufactur-
er’s software. For each run, a standard curve was gener-
ated from duplicate samples at different dilutions whose
copy numbers were known, and the RNA in the plasma
samples were quantified based on the copy number of
the standard samples.
Measurement of antibodies to HIV-1 Env protein in
plasma. HIV-1 Env-specific IgG antibodies in the plas-
ma were detected by ELISA using the following
method developed at Japan Immunoresearch Laborato-
ries, Takasaki, Japan (2). In brief, a 96-well microplate
(Nunc-ImmunoTMModules, MaxisorpTM, Nalge-Nunc,
Rochester, N.Y., U.S.A.) was coated with HIV-1 IIIB
gp160 recombinant viral protein (Advanced Biotech-
nologies, Inc., Columbia, Md., U.S.A.) at 1 µg/ml in
0.1 M Na2CO3-NaHCO3 buffer (pH 9.6), incubated
overnight at 4 C, washed with 0.15 M NaCl containing
0.05% Tween 20 and treated with 25% Block AceTM
(Nacalai Tesque, Inc., Kyoto, Japan) for 2 hr at room
temperature, incubated overnight with plasma diluted
with staining buffer (10% Block Ace) at 4 C, washed,
incubated with peroxidase-conjugated goat anti-monkey
IgG (1 µg/ml) (Kirkegaard & Perry Laboratories, Inc.,
Gaithersburg, Nd., U.S.A.) for 2 hr at room tempera-
ture, washed, treated with O-phenylenediamine dihy-
drochloride (OPD, Sigma, St. Louis, Mo., U.S.A.) for
10 min and immersed in 2 N H2SO4to stop the reaction.
Specific absorbances were read at 490 nm with a
microplate reader as described (2). The amounts of
antibody were expressed as the optical density. Plas-
mas of SHIV-infected monkeys and naïve monkeys
were used as positive and negative controls, respectively.
IFN-γ ELISPOT assay. The number of antigen spe-
cific cytokine rIFN-γ-producing cells was determined
with an IFN-γ ELISPOT kit (Mabtech, Nacka, Sweden)
using an MAHAS4510 multi-screen 96-well plate (Mil-
lipore, Bedford, Mass., U.S.A.), anti-monkey IFN-γ
antibody, biotinylated anti-IFN-γ antibody and strepta-
vidin-alkaline phosphatase conjugate antibody, accord-
ing to the manufacturer’s instructions. SIV Gag p27
protein (soluble native p27, Advanced biotechnologies)
was added to the cultures at a final concentration of 10
µg/ml. Medium alone was used as a negative control.
As a positive control, concanavalin A was added to
control wells at a final concentration of 5 µg/ml. Cells
were tested at 1?106cells per well in duplicate and
incubated undisturbed at 37 C, 5% CO2, for 36 hr. The
resulting spots were counted with a microscope. Date in
the figures show the mean number of spot-forming cells
(SFC) per 1?106PBMCs.
Flow cytometry. An absolute cell count was deter-
mined from samples of PBMCs as described previously
(13). PBMCs of each monkey were stained with FITC-
conjugated anti-monkey CD3 (FN-18; BioSource Inter-
national, Belgium), phycoerythrin (PE)-conjugated anti-
human CD4 (NU-TH/I; Nichirei), PerCP-conjugated
anti-human CD8 (leu-2a; BD Pharmingen, San Diego,
Calif., U.S.A.) or FITC-conjugated anti-human CD29
(4B4; Beckman Coulter). After hemolysis of the whole
blood using FACS TM Lysing Solution (BD Pharmin-
gen), each type of labeled lymphocytes was measured
on a FACScan (Becton Dickinson, Mountain View,
Calif., U.S.A.) by using Cell Quest software (Becton
Dickinson). Absolute lymphocyte counts in the blood
were determined with an automated blood cell counter
(F-820; Sysmex, Japan).
NK cytotoxicity assay. NK cell activities of PBMCs
in each monkey were periodically measured by a
chromium (51Cr) release assay using K562 cells as target
cells (12). Briefly, PBMCs obtained from each mon-
PROTECTIVE EFFICACY OF INF-γ AGAINST SHIV INFECTION
Table 1. Immunization and treatment
348, 352, 295
371, 291, 413, 414
367, 369, 405, 411, 412
a)1?106U, i.m. three times per week for 4 weeks.
key were used as effector cells. Serial dilutions of
effector cells were cultured with 1?104cells of 51Cr-
labeled K562 cells dispensed in triplicate into each well
of 96-well plates. After 4 hr, the culture supernatants of
each well were monitored with a γ counter. Cytotoxic
activity was calculated as the percent specific lysis at
an effector cells to target cells ratio of 50:1. Percent
specific lysis was calculated from (experimental
release?spontaneous release)/(total release?sponta-
neous release)?100. Total release was determined by
treating the target cells with 2.5% Triton X-100.
Measurement of cytokines and CC-chemokines.
PBMCs obtained from each monkey were incubated in
a tissue-culture dish for 1 hr and were separated into
adherent and non-adherent cells. We used peripheral
blood lymphocytes (PBLs), prepared as non-adherent
cells, in the cytokine and CC-chemokine detection
assays. After the stimulation of non-adherent cells with
the SIV Gag p27 protein (Advanced Biotechnologies) at
a final concentration of 10 µg/ml for 72 hr, the produc-
tions of IFN-γ, IL-4, IL-10, MIP-1α, MIP-1β,
RANTES were measured in the culture supernatants
using an immunoassay kit (BioSource International)
and Quantikine (R & D Systems, Minneapolis, Minn.,
No Antiviral Effect of rIFN-γ Administration to Unvac-
cinated Naïve Monkeys against SHIV-C2/1 Infection
First, to evaluate the antiviral effects of rIFN-γ with-
out vaccination against a challenge virus, SHIV-C2/1,
three of five unvaccinated monkeys (MM348, MM352
and MM295) were administered with rIFN-γ (group A)
and the other two (MM316 and MM328) were adminis-
tered with placebo (group B). rIFN-γ was administered
at a dose of 1?106U per monkey three times per week
for 4 weeks. Then these monkeys were challenged
intravenously with 200 TCID50 of a pathogenic SHIV-
C2/1. The five challenged monkeys were monitored for
viral load for 15 weeks.
As shown in Fig. 1-A, in placebo-administered naïve
monkeys (group B) that were infected with SHIV-C2/1,
the viral RNA loads in plasma dramatically increased to
about 6.3?107to 8.7?108at 2 weeks post challenge
(w.p.c.). On the other hand, in rIFN-γ-administered
monkeys (group A), the viral RNA loads in plasma
increased to about 1.6?107at 2 w.p.c. Thus, the peaks
of plasma viral load were reduced about 5-fold in group
A monkeys with rIFN-γ compared with the levels in
naïve group B monkeys. In both group A and group B,
almost all monkeys developed a rapid and complete
loss of CD4?T cells, but only one monkey in group A
(MM 295) did not show complete loss of CD4?T cells.
Viral set point levels of 4.6?106to 5.0?105copies/ml
were maintained in these unvaccinated monkeys. These
results show that rIFN-γ administration had little or no
Enhanced Protective Effect of rIFN-γ Administration to
Vaccinated Monkeys against SHIV-C2/1 Replication
To investigate the adjuvant effect of IFN-γ, nine
monkeys were immunized with SHIV-NI. Four of them
(MM371, MM291, MM413 and MM414) were admin-
istered with rIFN-γ (group C) and the other five mon-
keys (MM367, MM369, MM405, MM411, MM412)
were administered with placebo (group D). The dose
and schedule of rIFN-γ administration were the same
with those of group A. Then these monkeys were chal-
lenged with SHIV-C2/1.
Neither the rIFN-γ administered monkeys (group C)
nor the placebo-administered monkeys (group D) expe-
rienced the severe CD4?T cell loss after SHIV-C2/1
challenge that occurred in the unvaccinated monkeys
(Fig. 2-B). However, the plasma viral RNA loads after
the challenge were different in the two groups. In one
rIFN-γ-administered monkey (MM413, group C), viral
loads were undetectable after the challenge. In the
three other rIFN-γ-administered monkeys (group C), the
viral RNA loads in plasma quantified by PCR specific
for a challenge SHIV-C2/1 were about 1?103to 2?105
at 2 w.p.c. (Fig. 1-B). On the other hand, the plasma
viral RNA loads increased to about 3?107to 1?106at 2
w.p.c. in placebo-administered monkeys (group D).
Thus, in the vaccinated monkeys with rIFN-γ (group
C), the peaks of plasma viral load were reduced 100- to
1,000-fold compared to those in the vaccinated mon-
keys without rIFN-γ (group D). The significant differ-
ence of the peak values of plasma viral load between
groups C and D showed that vaccination of SHIV-NI in
combination with rIFN-γ administration could induce
strong resistance to SHIV-C2/1 replication.
Enhancement of IFN-γ Producing Cells in the Vaccinat-
ed Monkeys with rIFN-γ
To investigate whether IFN-γ augments the immune
response induced by vaccination with a live attenuated
SHIV, the number of virus specific spot forming cells
(SFC) secreting IFN-γ of PBMCs in each monkey
groups were measured by ELISPOT assays.
SIV Gag-specific SFC induced in the SHIV-NI vacci-
nated monkeys receiving rIFN-γ (group C) was higher
than that induced in the vaccinated monkeys without
rIFN-γ. Two weeks after the challenge, group C with
rIFN-γ reached the highest number of Gag-specific
IFN-γ SFC (Fig. 3-B). On the other hand, in the unvac-
K. KANEYASU ET AL
cinated monkeys with and without rIFN-γ (groups A
and B), the number of Gag-specific IFN-γ SFC were
hardly detectable (Fig. 3-A). These results demonstrat-
ed that vaccination of SHIV-NI in combination with
rIFN-γ administration augments the cellular immune
Delayed Antibody Response in the SHIV-NI Vaccinated
Monkeys Receiving rIFN-γ
The time of appearance of HIV-1 Env-specific IgG
antibodies detected by ELISA was different in vaccinat-
ed macaques with and without rIFN-γ after the chal-
lenge (Fig. 4). The SHIV-NI-vaccinated monkeys that
received the placebo (group D) exhibited HIV-1 gp160-
specific IgG antibody responses gradually from 1 w.p.c.
On the other hand, at 1 w.p.c. no antibody was detected
in the SHIV-NI vaccinated monkeys that received rIFN-
γ (MM291, MM413 and MM414) (group C). At 2
w.p.c. the titer of antibody dramatically increased in
both monkey groups but was slightly higher in group D
without rIFN-γ than in group C with rIFN-γ, which
showed a higher Th1 response as shown in the previous
section. No HIV-1 Env-specific IgG antibodies were
detected in any of the vaccinated macaques before chal-
lenge (the cut off value was 0.11).
Enhanced Chemokine MIP-1α Production in the SHIV-
NI Vaccinated Monkeys Receiving rIFN-γ
To assess the production of CC-hemokines and
cytokines by antigen-stimulated PBMCs from the vacci-
PROTECTIVE EFFICACY OF INF-γ AGAINST SHIV INFECTION
(B) Vaccinated monkeys.
Fig. 1. Kinetics of plasma viral RNA of the SHIV C2/1-infected monkeys. (A) Unvaccinated monkeys;
(B) vaccinated monkeys. The experimental schedule of each group of monkeys is presented in Table 1.
(A) Unvaccinated monkeys.
nated monkeys (groups C and D), the production of
MIP-1α, MIP-1β, RANTES, IFN-γ, IL-4 and IL-10 by
specific ELISAs was analyzed. PBMCs of nine vacci-
nated monkeys (groups C and D) were stimulated with
SIV Gag and their presence in cell culture supernatants
were measured before and at 2 weeks after vaccination.
No substantial differences were seen in the produc-
tion of MIP-1β, RANTES, IFN-γ, IL-4 or IL-10 by
PBMCs stimulated with SIV Gag in the SHIV-NI vacci-
nated monkeys with and without rIFN-γ during the time
of follow-up (data not shown). However, the levels of
MIP-1α secreted by PBMCs in SHIV-NI-vaccinated
monkeys receiving rIFN-γ (group C) were dramatically
higher at 2 week post vaccination (w.p.v.), than those of
vaccinated monkeys without rIFN-γ (group D) (Fig. 5).
Augmentation of NK Activity by rIFN-γ Administration
To evaluate innate immune response that contributed
to the protection against SHIV-C2/1, we assayed NK
cell activity of PBMCs of the SHIV-NI vaccinated
monkeys with (group C) and without (group D) rIFN-γ.
In all the group C monkeys except MM291, NK cell
K. KANEYASU ET AL
(B) Vaccinated monkeys.
Fig. 2. Changes in CD4?T cell counts in PBMCs of unvaccinated monkeys (A) and
SHIV-NI-vaccinated monkeys (B) after SHIV C2/1 challenge.
(A) Unvaccinated monkeys.
activities at 1 w.p.v. were approximately 30 to 45%
greater than the activities before rIFN-γ treatment (Fig.
6). These augmented NK activities were observed dur-
ing the 3-week rIFN-γ treatment period. On the other
hand, no augmentation was observed in the vaccinated
monkeys without IFN-γ (group D). After the challenge,
The NK activities of these monkeys remained at low
levels in both groups. These results demonstrate that
rIFN-γ administration augmented NK cell activities in
the vaccinated monkeys.
We previously reported that monkeys immunized
with a nef-deleted SHIV-NM-3rN (SHIV-NI) or an
SHIV-NI expressing human IFN-γ (SHIV-IFN-γ) could
control the replication of a heterologous pathogenic
virus and prevent the loss of CD4?T cells. SHIV-IFN-γ
vaccinated monkeys showed resistance to the SHIV
C2/1 challenge, even though only 4 weeks had passed
PROTECTIVE EFFICACY OF INF-γ AGAINST SHIV INFECTION
(B) Vaccinated monkeys.
Fig. 3. Numbers of SIV Gag-specific IFN-γ-producing cells in PBMCs of unvaccinated
monkeys (A) and vaccinated monkeys (B). PBMCs were stimulated with SIV Gag protein.
IFN-γ-producing cells were detected by IFN-γ-specific ELISPOT assays, and data are
expressed as the number of SFC per 106cells. Each point represents the mean number of
IFN-γ spots in duplicate wells.
(A) Unvaccinated monkeys.
since the immunization. Moreover, the peak value of
the plasma viral load in the early phase of the challenge
with SHIV-C2/1 was reduced in the SHIV-IFN-γ-vacci-
nated monkeys compared with those in the SHIV-NI
vaccinated monkeys. These results raise the possibility
that IFN-γ makes a strong contribution to the suppres-
sion of SHIV-C2/1 replication (13, 23). The other
study has been reported that HIV p24 antigen was
decreased in plasma samples obtained from six of nine
rIFN-γ-administered patients with initially detectable
HIV protein (19). These data suggest that rIFN-γ
should be considered as a therapeutic agent, possibly
with other antiviral, in the treatment of patients with
AIDS. However, whether exogenous IFN-γ administra-
tion induces protective immunity in the SHIV/monkey
model which is useful for clarifying the mechanism of
IFN-γ-induced protection is unknown.
The aim of the present study was to investigate
whether exogenous IFN-γ administration could aug-
ment the immune response induced by vaccination with
a live attenuated SHIV-NI. In the vaccinated monkey
groups, SHIV-NI vaccination combined with rIFN-γ
administration resulted in significantly limiting the peak
value of the plasma viral load and maintaining CD4?T
cells after SHIV-C2/1 challenge. In this group, the
peak of plasma viral load was reduced approximately
100- to 1,000-fold and the number of SIV Gag-specific
IFN-γ-producing cells increased 2-fold compared with
those in the SHIV-NI-vaccinated monkeys without
rIFN-γ (group D). On the other hand, in the unvaccinat-
K. KANEYASU ET AL
Fig. 4. HIV-1 Env-specific IgG antibodies in plasma of SHIV-NI vaccinated monkeys combined with rIFN-γ or
placebo administration after challenge.
Fig. 5. Production of MIP-1α by SIV Gag-stimulated PBMCs of SHIV-NI vaccinated monkeys combined with
rIFN-γ or placebo. PBMCs were obtained prior to vaccination (0) and 2 weeks after vaccination.
ed monkey groups, CD4?T cells declined after SHIV-
C2/1 infection almost equally in monkeys with rIFN-γ
(group A) and without (group B). SIV Gag-specific
IFN-γ-producing cells were undetectable in unvaccinat-
ed monkeys of both groups. The increase in the number
of IFN-γ-producing cells in the vaccinated monkeys
was closely correlated with the decrease in the peak
value of plasma viral load of SHIV-C2/1.
These data suggest that vaccination with SHIV-NI in
combination with rIFN-γ administration is more effec-
tive at modulating the immune system into the Th1-type
response than either rIFN-γ administration alone or
SHIV-NI vaccination alone. This modulation to a Th1-
type response indicates that rIFN-γ administration could
induce the strong cellular immune response against
SHIV infection. The augmented Th1-type responses
assessed by SIV Gag protein-specific IFN-γ-producing
cell activity might be associated with the activity of
cytotoxic T lymphocytes (CTLs). Antigen-specific
Th1-mediated immune responses and CTLs have been
reported to provide protection and reduce disease pro-
gression (16, 22, 32, 36, 39). Th1/CD8?T cell responses
have been shown to play an important role in controlling
HIV-1 replication (5, 27, 34, 35, 37, 38, 40, 42–44).
Th1/CD4?T cell responses (antigen-specific CD4?T
helper cells) also may promote CTL activity either by a
CD4-antigen-presenting cell (APC)-CD8 pathway or by
IL-2 secretion (20, 41, 57). The present observations
are encouraging in light of the hypothesis that Th1-
mediated immunity is associated with resistance to
virus infection and suppression (3, 28).
To investigate whether IFN-γ affect the innate immu-
nity, we examined production of CC-chemokines and
cytokines, and the NK activity of freshly isolated
PBMCs of these monkeys. The levels of RANTES,
MIP-1α, MIP-1β, IFN-γ, IL-4 and IL-10 of super-
natants from SIV Gag-stimulated PBMCs were mea-
sured by ELISA. In result of these, NK activity and
MIP-1α production were enhanced in SHIV-NI-vacci-
nated monkeys with rIFN-γ (group C). Moreover, no
HIV-1 Env-specific antibody was detected at 1 w.p.c. in
SHIV-NI-vaccinated monkeys with rIFN-γ. On the
other hand, SHIV-NI vaccinated monkeys without
rIFN-γ (group D) exhibited HIV-1 Env-specific anti-
body responses at 1 w.p.c. The lower IgG Ab response
generated in SHIV-NI vaccinated monkeys with rIFN-γ
might have resulted from the predominant Th1?Th2
cytokine response modulated probably by both NK
activity and MIP-1α production. Several studies have
shown that MIP-1α stimulation enhances IFN-γ pro-
duction, which is essential for the induction of Th1-
derived HIV-specific cell-mediated immunity (25).
MIP-1α was reported to activate NK cells and the acti-
vated NK cells produce MIP-1α (29, 31, 45). NK cells
are a critical component of the host innate immune
response to a variety of viruses, fungi, parasites and
bacteria (35, 45, 52). After activation, NK cells release
various cytokines and chemokines including MIP-1α
that induce the inflammatory response, modulate
hematopoiesis, control monocyte and granulocyte
growth and function, and influence the type of adaptive
immune responses (52). With regard to HIV infection,
the nonspecificity of NK cell activity might be relevant
to the maintenance of a degree of antiviral activity in the
PROTECTIVE EFFICACY OF INF-γ AGAINST SHIV INFECTION
Fig. 6. NK cell activity of PBMCs in the SHIV-NI vaccinated monkeys combined with rIFN-γ or
placebo administration before and after challenge.
face of a high level of virus replication that negatively
impacts HIV-specific cellular and humoral immune
responses (7, 15). Recently, Enose et al. (13) showed
vaccination of monkeys with live attenuated SHIV hav-
ing IFN-γ effectively suppressed the peak value of plas-
ma viral loads from 1 to 3 weeks after SHIV-C2/1 chal-
lenge. Cellular immune responses were augmented by
SHIV-IFN-γ as compared to SHIV-NI without the IFN-γ
gene insert. From 4 to 12 weeks after the challenge,
however, these monkeys showed a transient increase in
viral load. IFN-γ mediated inflammation has been
reported to be associated with a lack of protection from
SIV challenge in vaccinated rhesus macaques (1). It is
possible that IFN-γ-driven inflammation promotes
SHIV-C2/1 replication in SHIV-IFN-γ immunized mon-
keys at later stage, because SHIV-IFN-γ immunization
may increase IFN-γ mRNA levels in lymphoid tissues
(1). In a vaccine study using a combination of DNA
immunization with plasmid adjuvants expressing GM-
CSF and IFN-γ (30), both humoral and cellular immune
responses to SIV antigens were augmented by cytokine
expression plasmids as compared to mock plasmid
without a cytokine gene insert, but the plasma viral
loads at set points were not significantly different
between both the immunized group and the non-immu-
nized control group. On the other hand, in this present
study, the SHIV-NI-vaccinated monkeys that were treat-
ed with rIFN-γ (group C) exhibited various responses
including augmentation of HIV-1 Env-specific antibody
responses. In addition, the innate and cellular immune
responses in these monkeys significantly reduced the
peak value of the plasma viral load without a transient
increase in viral RNA load after the challenge. To
obtain better protection against a pathogenic virus, it
may be necessary to develop a vaccine that induces
both innate and cellular immune responses.
In conclusion, our data show that SHIV-NI vaccina-
tion combined with rIFN-γ administration induces
strong SIV Gag-specific Th1-type cellular immune
response, which might contribute to the control of a
heterologous pathogenic SHIV challenge infection.
Since the magnitude of the induced immunity is consid-
ered enough for protection against viral infection and
disease progression, rIFN-γ could be used as an adjuvant
for attenuated vaccine candidates against SHIV infec-
tion, probably against HIV infection.
We thank James Raymond for English editing of this manu-
script. We are grateful to Ms. Ai Himeno and Ms. Humiko
Ogatu for technical assistance. This work was supported by a
Grant-in-Aid for Scientific Research from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan and a
Research Grant on Health Sciences focusing on Drug Innovation
from the Japan Health Sciences Foundation. K.K. is supported
by the 21st Century COE Program of the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
1) Abel, K., La Franco-Scheuch, L., Rourke, T., Ma, Z.M., De
Silva, V., Fallert, B., Beckett, L., Reinhart, T.A., and Miller,
C.J. 2004. Gamma interferon-mediated inflammation is
associated with lack of protection from intravaginal simian
immunodeficiency virus SIVmac239 challenge in simian-
human immunodeficiency virus 89.6-immunized rhesus
macaques. J. Virol. 78: 841–854.
2) Akagi, T., Kawamura, M., Ueno, M., Hiraishi, K., Adachi,
M., Serizawa, T., Akashi, M., and Baba, M. 2003. Mucosal
immunization with inactivated HIV-1-capturing nano-
spheres induces a significant HIV-1-specific vaginal anti-
body response in mice. J. Med. Virol. 69: 163–172.
3) Bailer, R.T., Holloway, A., Sun, J., Margolick, J.B., Martin,
M., Kostman, J., and Montaner, L.J. 1999. IL-13 and IFN-
gamma secretion by activated T cells in HIV-1 infection
associated with viral suppression and a lack of disease pro-
gression. J. Immunol. 162: 7534–7542.
4) Benito, J.M., Lopez, M., and Soriano, V. 2004. The role of
CD8? T-cell response in HIV infection. AIDS Rev. 6:
5) Bernard, N.F., Yannakis, C.M., Lee, J.S., and Tsoukas,
C.M. 1999. Human immunodeficiency virus (HIV)-specific
cytotoxic T lymphocyte activity in HIV-exposed seronega-
tive persons. J. Infect. Dis. 179: 538–547.
6) Billiau, A. 1998. Interferon-gamma, the TH1/TH2 para-
digm in autoimmunity. Bull. Mem. Acad. R. Med. Belg.
7) Bluman, E.M., Bartynski, K.J., Avalos, B.R., and Caligiuri,
M.A. 1996. Human natural killer cells produce abundant
macrophage inflammatory protein-1 alpha in response to
monocyte-derived cytokines. J. Clin. Invest. 97:
8) Chatterjee, S., and Hunter, E. 1987. Recombinant human
interferons inhibit replication of Mason-Pfizer monkey
virus in primate cells. Virology 157: 548–551.
9) Connor, R.I., Montefiori, D.C., Binley, J.M., Moore, J.P.,
Bonhoeffer, S., Gettie, A., Fenamore, E.A., Sheridan, K.E.,
Ho, D.D., Dailey, P.J., and Marx, P.A. 1998. Temporal
analyses of virus replication, immune responses, and effica-
cy in rhesus macaques immunized with a live, attenuated
simian immunodeficiency virus vaccine. J. Virol. 72:
10) Constantoulakis, P., Campbell, M., Felber, B.K., Nasioulas,
G., Afonina, E., and Pavlakis, G.N. 1993. Inhibition of
Rev-mediated HIV-1 expression by an RNA binding pro-
tein encoded by the interferon-inducible 9-27 gene. Science
11) Daniel, M.D., Kirchhoff, F., Czajak, S.C., Sehgal, P.K., and
Desrosiers, R.C. 1992. Protective effects of a live attenuated
SIV vaccine with a deletion in the nef gene. Science 258:
12) De Maria, A., Fogli, M., Costa, P., Murdaca, G., Puppo, F.,
Mavilio, D., Moretta, A., and Moretta, L. 2003. The
K. KANEYASU ET AL
impaired NK cell cytolytic function in viremic HIV-1 infec-
tion is associated with a reduced surface expression of nat-
ural cytotoxicity receptors (NKp46, NKp30 and NKp44).
Eur. J. Immunol. 33: 2410–2418.
13) Enose, Y., Kita, M., Yamamoto, T., Suzuki, H., Miyake, A.,
Horiuchi, R., Ibuki, K., Kaneyasu, K., Kuwata, T., Taka-
hashi, E., Sakai, K., Shinohara, K., Miura, T., and Hayami,
M. 2004. Protective effects of nef-deleted SHIV or that
having IFN-gamma against disease induced with a patho-
genic virus early after vaccination. Arch. Virol. 149:
14) Enose, Y., Ui, M., Miyake, A., Suzuki, H., Uesaka, H.,
Kuwata, T., Kunisawa, J., Kiyono, H., Takahashi, H.,
Miura, T., and Hayami, M. 2002. Protection by intranasal
immunization of a nef-deleted, nonpathogenic SHIV
against intravaginal challenge with a heterologous patho-
genic SHIV. Virology 298: 306–316.
15) Fehniger, T.A., Herbein, G., Yu, H., Para, M.I., Bernstein,
Z.P., O’Brien, W.A., and Caligiuri, M.A. 1998. Natural
killer cells from HIV-1? patients produce C-C chemokines
and inhibit HIV-1 infection. J. Immunol. 161: 6433–6438.
16) Ferrari, C., Penna, A., Bertoletti, A., Cavalli, A., Missale,
G., Lamonaca, V., Boni, C., Valli, A., Bertoni, R., Urbani,
S., Scognamiglio, P., and Fiaccadori, F. 1998. Antiviral cell-
mediated immune responses during hepatitis B and hepatitis
C virus infections. Recent Results Cancer. Res. 154:
17) Giavedoni, L., Ahmad, S., Jones, L., and Yilma, T. 1997.
Expression of gamma interferon by simian immunodefi-
ciency virus increases attenuation and reduces postchal-
lenge virus load in vaccinated rhesus macaques. J. Virol.
18) Hartshorn, K.L., Neumeyer, D., Vogt, M.W., Schooley,
R.T., and Hirsch, M.S. 1987. Activity of interferons alpha,
beta, and gamma against human immunodeficiency virus
replication in vitro. AIDS Res. Hum. Retroviruses 3:
19) Heagy, W., Groopman, J., Schindler, J., and Finberg, R.
1990. Use of IFN-gamma in patients with AIDS. J. Acquir.
Immune Defic. Syndr. 3: 584–590.
20) Heeney, J.L. 2002. The critical role of CD4 (?) T-cell help
in immunity to HIV. Vaccine 20: 1961–1963.
21) Huang, S., Hendriks, W., Althage, A., Hemmi, S., Blueth-
mann, H., Kamijo, R., Vilcek, J., Zinkernagel, R.M., and
Aguet, M. 1993. Immune response in mice that lack the
interferon-gamma receptor. Science 259: 1742–1745.
22) Hukkanen, V., Broberg, E., Salmi, A., and Eralinna, J.P.
2002. Cytokines in experimental herpes simplex virus
infection. Int. Rev. Immunol. 21: 355–371.
23) Iida, T., Kuwata, T., Ui, M., Suzuki, H., Miura, T., Ibuki, K.,
Takahashi, H., Yamamoto, T., Imanishi, J., Hayami, M., and
Kita, M. 2004. Augmentation of antigen-specific cytokine
responses in the early phase of vaccination with a live-
attenuated simian/human immunodeficiency chimeric virus
expressing IFN-gamma. Arch. Virol. 149: 743–757.
24) Johnson, R.P., Lifson, J.D., Czajak, S.C., Cole, K.S., Man-
son, K.H., Glickman, R., Yang, J., Montefiori, D.C., Monte-
laro, R., Wyand, M.S., and Desrosiers, R.C. 1999. Highly
attenuated vaccine strains of simian immunodeficiency
virus protect against vaginal challenge: inverse relationship
of degree of protection with level of attenuation. J. Virol. 73:
25) Karpus, W.J., Lukacs, N.W., Kennedy, K.J., Smith, W.S.,
Hurst, S.D., and Barrett, T.A. 1997. Differential CC
chemokine-induced enhancement of T helper cell cytokine
production. J. Immunol. 158: 4129–4136.
26) Karupiah, G., Blanden, R.V., and Ramshaw, I.A. 1990.
Interferon gamma is involved in the recovery of athymic
nude mice from recombinant vaccinia virus/interleukin 2
infection. J. Exp. Med. 172: 1495–1503.
27) Kaul, R., Rowland-Jones, S.L., Kimani, J., Fowke, K.,
Dong, T., Kiama, P., Rutherford, J., Njagi, E., Mwangi, F.,
Rostron, T., Onyango, J., Oyugi, J., MacDonald, K.S.,
Bwayo, J.J., and Plummer, F.A. 2001. New insights into
HIV-1 specific cytotoxic T-lymphocyte responses in
exposed, persistently seronegative Kenyan sex workers.
Immunol. Lett. 79: 3–13.
28) Kostense, S., Vandenberghe, K., Joling, J., Van Baarle, D.,
Nanlohy, N., Manting, E., and Miedema, F. 2002. Persis-
tent numbers of tetramer? CD8 (?) T cells, but loss of
interferon-gamma? HIV-specific T cells during progres-
sion to AIDS. Blood 99: 2505–2511.
29) Kottilil, S., Chun, T.W., Moir, S., Liu, S., McLaughlin, M.,
Hallahan, C.W., Maldarelli, F., Corey, L., and Fauci, A.S.
2003. Innate immunity in human immunodeficiency virus
infection: effect of viremia on natural killer cell function. J.
Infect. Dis. 187: 1038–1045.
30) Lena, P., Villinger, F., Giavedoni, L., Miller, C.J., Rhodes,
G., and Luciw, P. 2002. Co-immunization of rhesus
macaques with plasmid vectors expressing IFN-gamma,
GM-CSF, and SIV antigens enhances anti-viral humoral
immunity but does not affect viremia after challenge with
highly pathogenic virus. Vaccine 20 (Suppl 4): A69–79.
31) Loetscher, P., Seitz, M., Clark-Lewis, I., Baggiolini, M.,
and Moser, B. 1996. Activation of NK cells by CC
chemokines. Chemotaxis, Ca2?mobilization, and enzyme
release. J. Immunol. 156: 322–327.
32) Lusso, P. 2000. Chemokines and viruses: the dearest ene-
mies. Virology 273: 228–240.
33) Mason, R.D., Bowmer, M.I., Howley, C.M., and Grant,
M.D. 2005. Cross-reactive cytotoxic T lymphocytes against
human immunodeficiency virus type 1 protease and gamma
interferon-inducible protein 30. J. Virol. 79: 5529–5536.
34) Musey, L., Hughes, J., Schacker, T., Shea, T., Corey, L., and
McElrath, M.J. 1997. Cytotoxic-T-cell responses, viral load,
and disease progression in early human immunodeficiency
virus type 1 infection. N. Engl. J. Med. 337: 1267–1274.
35) Ogg, G.S., Jin, X., Bonhoeffer, S., Dunbar, P.R., Nowak,
M.A., Monard, S., Segal, J.P., Cao, Y., Rowland-Jones,
S.L., Cerundolo, V., Hurley, A., Markowitz, M., Ho, D.D.,
Nixon, D.F., and McMichael, A.J. 1998. Quantitation of
HIV-1-specific cytotoxic T lymphocytes and plasma load of
viral RNA. Science 279: 2103–2106.
36) Ohga, S., Nomura, A., Takada, H., and Hara, T. 2002.
Immunological aspects of Epstein-Barr virus infection. Crit.
Rev. Oncol. Hematol. 44: 203–215.
37) Pinto, L.A., Sullivan, J., Berzofsky, J.A., Clerici, M.,
Kessler, H.A., Landay, A.L., and Shearer, G.M. 1995. ENV-
PROTECTIVE EFFICACY OF INF-γ AGAINST SHIV INFECTION
specific cytotoxic T lymphocyte responses in HIV seroneg- Download full-text
ative health care workers occupationally exposed to HIV-
contaminated body fluids. J. Clin. Invest. 96: 867–876.
38) Pontesilli, O., Klein, M.R., Kerkhof-Garde, S.R., Pakker,
N.G., de Wolf, F., Schuitemaker, H., and Miedema, F. 1998.
Longitudinal analysis of human immunodeficiency virus
type 1-specific cytotoxic T lymphocyte responses: a pre-
dominant gag-specific response is associated with nonpro-
gressive infection. J. Infect. Dis. 178: 1008–1018.
39) Rico, M.A., Quiroga, J.A., Subira, D., Castanon, S., Este-
ban, J.M., Pardo, M., and Carreno, V. 2001. Hepatitis B
virus-specific T-cell proliferation and cytokine secretion in
chronic hepatitis B antibody-positive patients treated with
ribavirin and interferon alpha. Hepatology 33: 295–300.
40) Rinaldo, C., Huang, X.L., Fan, Z.F., Ding, M., Beltz, L.,
Logar, A., Panicali, D., Mazzara, G., Liebmann, J., Cottrill,
M., et al. 1995. High levels of anti-human immunodeficien-
cy virus type 1 (HIV-1) memory cytotoxic T-lymphocyte
activity and low viral load are associated with lack of dis-
ease in HIV-1-infected long-term nonprogressors. J. Virol.
41) Rosenberg, E.S., Billingsley, J.M., Caliendo, A.M.,
Boswell, S.L., Sax, P.E., Kalams, S.A., and Walker, B.D.
1997. Vigorous HIV-1-specific CD4? T cell responses
associated with control of viremia. Science 278:
42) Rowland-Jones, S.L., and McMichael, A. 1995. Immune
responses in HIV-exposed seronegatives. Curr. Opin.
Immunol. 7: 448–455.
43) Rowland-Jones, S.L., Nixon, D.F., Aldhous, M.C., Gotch,
F., Ariyoshi, K., Hallam, N., Kroll, J.S., Froebel, K., and
McMichael, A. 1993. HIV-specific cytotoxic T-cell activity
in an HIV-exposed but uninfected infant. Lancet 341:
44) Rowland-Jones, S., Sutton, J., Ariyoshi, K., Dong, T.,
Gotch, F., McAdam, S., Whitby, D., Sabally, S., Gallimore,
A., Corrah, T., et al. 1995. HIV-specific cytotoxic T-cells in
HIV-exposed but uninfected Gambian women. Nat. Med. 1:
45) Salazar-Mather, T.P., Hamilton, T.A., and Biron, C.A. 2000.
A chemokine-to-cytokine-to-chemokine cascade critical in
antiviral defense. J. Clin. Invest. 105: 985–993.
46) Schmitz, J.E., Kuroda, M.J., Santra, S., Sasseville, V.G.,
Simon, M.A., Lifton, M.A., Racz, P., Tenner-Racz, K.,
Dalesandro, M., Scallon, B.J., Ghrayeb, J., Forman, M.A.,
Montefiori, D.C., Rieber, E.P., Letvin, N.L., and Reimann,
K.A. 1999. Control of viremia in simian immunodeficiency
virus infection by CD8? lymphocytes. Science 283:
47) Shinohara, K., Sakai, K., Ando, S., Ami, Y., Yoshino, N.,
Takahashi, E., Someya, K., Suzaki, Y., Nakasone, T., Sasaki,
Y., Kaizu, M., Lu, Y., and Honda, M. 1999. A highly patho-
genic simian/human immunodeficiency virus with genetic
changes in cynomolgus monkey. J. Gen. Virol. 80:
48) Sivori, S., Pende, D., Bottino, C., Marcenaro, E., Pessino,
A., Biassoni, R., Moretta, L., and Moretta, A. 1999. NKp46
is the major triggering receptor involved in the natural cyto-
toxicity of fresh or cultured human NK cells. Correlation
between surface density of NKp46 and natural cytotoxicity
against autologous, allogeneic or xenogeneic target cells.
Eur. J. Immunol. 29: 1656–1666.
49) Stahl-Hennig, C., Gundlach, B.R., Dittmer, U., ten Haaft, P.,
Heeney, J., Zou, W., Emilie, D., Sopper, S., and Uberla, K.
2003. Replication, immunogenicity, and protective proper-
ties of live-attenuated simian immunodeficiency viruses
expressing interleukin-4 or interferon-gamma. Virology
50) Suryanarayana, K., Wiltrout, T.A., Vasquez, G.M., Hirsch,
V.M., and Lifson, J.D. 1998. Plasma SIV RNA viral load
determination by real-time quantification of product genera-
tion in reverse transcriptase-polymerase chain reaction.
AIDS Res. Hum. Retroviruses 14: 183–189.
51) Taylor, M.D., Korth, M.J., and Katze, M.G. 1998. Interferon
treatment inhibits the replication of simian immunodefi-
ciency virus at an early stage: evidence for a block between
attachment and reverse transcription. Virology 241:
52) Trinchieri, G. 1989. Biology of natural killer cells. Adv.
Immunol. 47: 187–376.
53) Turner, P.K., Houghton, J.A., Petak, I., Tillman, D.M.,
Douglas, L., Schwartzberg, L., Billups, C.A., Panetta, J.C.,
and Stewart, C.F. 2004. Interferon-gamma pharmacokinetics
and pharmacodynamics in patients with colorectal cancer.
Cancer Chemother. Pharmacol. 53: 253–260.
54) Ui, M., Kuwata, T., Igarashi, T., Ibuki, K., Miyazaki, Y.,
Kozyrev, I.L., Enose, Y., Shimada, T., Uesaka, H.,
Yamamoto, H., Miura, T., and Hayami, M. 1999. Protec-
tion of macaques against a SHIV with a homologous HIV-1
Env and a pathogenic SHIV-89.6P with a heterologous Env
by vaccination with multiple gene-deleted SHIVs. Virology
55) Ui, M., Kuwata, T., Igarashi, T., Miyazaki, Y., Tamaru, K.,
Shimada, T., Nakamura, M., Uesaka, H., Yamamoto, H.,
and Hayami, M. 1999. Protective immunity of gene-deleted
SHIVs having an HIV-1 Env against challenge infection
with a gene-intact SHIV. J. Med. Primatol. 28: 242–248.
56) Winders, B.R., Schwartz, R.H., and Bruniquel, D. 2004. A
distinct region of the murine IFN-gamma promoter is
hypomethylated from early T cell development through
mature naive and Th1 cell differentiation, but is hyper-
methylated in Th2 cells. J. Immunol. 173: 7377–7384.
57) Wodarz, D., and Jansen, V.A. 2001. The role of T cell help
for anti-viral CTL responses. J. Theor. Biol. 211: 419–432.
K. KANEYASU ET AL