JOURNAL OF VIROLOGY, Aug. 2005, p. 10386–10396
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 16
Influence of Glycosylation on the Efficacy of an Env-Based Vaccine
against Simian Immunodeficiency Virus SIVmac239 in a
Macaque AIDS Model
Kazuyasu Mori,1,2,3* Chie Sugimoto,1,2,3Shinji Ohgimoto,4Emi E. Nakayama,5Tatsuo Shioda,5
Shigeru Kusagawa,1Yutaka Takebe,1Munehide Kano,1Tetsuro Matano,6Takae Yuasa,7
Daisuke Kitaguchi,7Masaaki Miyazawa,7Yumiko Takahashi,8Michio Yasunami,8
Akinori Kimura,8Naoki Yamamoto,1Yasuo Suzuki,3,9
and Yoshiyuki Nagai10
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640,1Tsukuba Primate Research
Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki 305-0843,2CREST, Japan Science and
Technology Agency, Kawaguchi, Saitama 332-0012,3Microbiology and Genomics, Department of Genome
Sciences, Kobe University School of Medicine, Kobe, Hyogo 650-0017,4Department of Viral Infections,
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871,5Department of
Microbiology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033,6
Department of Immunology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511,7
Department of Molecular Pathogenesis, Division of Medical Science, Medical Research Institute,
Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062,8Department of
Biochemistry, University of Shizuoka School of Pharmaceutical Sciences and
COE Program in the 21st Century, Shizuoka, Shizuoka 422-8526,9and
Toyama Institute of Health, Kosugi, Toyama 939-0363,10Japan
Received 8 December 2004/Accepted 2 May 2005
The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency
viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient
immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple
deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (?5G). In
?5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies
suggested that an understanding of the role of glycosylation is critical in defining not only the virological
properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the
development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines
consisting of Env from SIVmac239 and ?5G and compared their immunogenicities and vaccine efficacies by
challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine
groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized
animals than in ?5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-
Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in
the chronic phase. Unexpectedly, viral loads in ?5G Env-immunized animals were indistinguishable from
those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against
homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune
responses and vaccine efficacy.
Primate lentiviruses, human immunodeficiency viruses
(HIVs), and simian immunodeficiency viruses (SIVs) share
common genetic and biological properties. As SIVmac, origi-
nally isolated from macaques in primate research centers in the
United States, causes AIDS in macaques with remarkable sim-
ilarities to HIV type 1 (HIV-1) infection in humans, this AIDS
monkey model has been utilized to study vaccine development
and the pathogenesis of HIV infection (for reviews, see refer-
ences 10, 14, 17, 43, and 47).
HIV/SIV infection in the host consists of two phases, the
primary infection and chronic infection. During the primary
infection, extensive viral replication and dissemination of the
infection occur. In chronic infection, viral replication continues
for a long period, eventually leading to AIDS. Due to the host
immune response against the infection, these two phases are
separated by a set point at which the viral load reaches its
lowest level. The viral loads of the set point and chronic infec-
tion are inversely correlated with the control of SIV/HIV in-
fection and predict disease progression (25, 31); however, it
remains unclear which host responses determine the viral loads
of the set point and chronic infection. Nevertheless, virus-
specific immune responses have been implicated in the host’s
control of the infection. Cellular immunity, such as that shown
by cytotoxic T lymphocytes (CTL) and helper T cells, has been
reported to correlate with the control of HIV/SIV infection
(for reviews, see references 2, 24, 28, and 39). The role of the
neutralizing antibody (NAb) in the control of infection and the
* Corresponding author. Mailing address: Tsukuba Primate Re-
search Center, National Institute of Biomedical Innovation, 1 Hachi-
mandai, Tsukuba, Ibaraki 305-0843, Japan. Phone: 81-29-837-2121.
Fax: 81-29-837-0218. E-mail: firstname.lastname@example.org.
emergence of escape mutants has also been reported previ-
ously (7, 16, 51).
Despite these immune responses against HIV/SIV infection,
humans and macaques fail to contain the infection due to the
virus properties. HIV/SIV infects major target cells, such as
CD4?T cells and macrophages, by binding viral envelope
glycoproteins (Env) to cellular surface proteins and CD4 and
chemokine receptors (CCR5, CXCR4, or others) on target
cells (5, 32). Since viral entry consists of multiple steps (virion
binding to these viral receptors, conformational change of Env,
and fusion between the virion and the cellular membrane) and
the critical parts of Env used in these steps are exposed only
during each step, naturally generated antibodies are only partly
effective in preventing HIV/SIV infection in their hosts (7, 8).
Primary isolates can be neutralized to various degrees by HIV-
infected patient serum but not by contemporaneous autolo-
gous samples. Consequently, escape mutants against preexist-
ing NAb are selectively replicated (51). Thus, effective NAb is
rarely induced in HIV/SIV infection (8, 10). This could partly
explain the failure of Env-based vaccine trials against HIV-1
The heavy glycosylation of Env is a unique feature of HIV/
SIV that is distinctive from features of other enveloped viruses
and is significantly related to their neutralization-resistant
property (8, 29, 44). We therefore assumed that the insufficient
immune containment of HIV/SIV might be due to heavy gly-
cosylation in Env and that the removal of some glycans might
allow the host to mount a protective immune response against
the infection. Thus, we studied the influence of deglycosylation
on the replication of SIVmac239 in a T-cell line and created a
quintuple deglycosylation mutant of SIVmac239 (?5G), which
has maximal removal of N-glycans at amino acid residues 79,
146, 171, 460, and 479 in Env and retains a replication capa-
bility similar to that of SIVmac239 in phytohemagglutinin-
stimulated rhesus peripheral blood mononuclear cells (PBMCs)
(36, 40). We then examined the infection of rhesus macaques
with ?5G; although ?5G was replicated as extensively as
SIVmac239 during the primary infection, the subsequent ?5G
infection was restricted to a level less than the detection sen-
sitivity of a plasma viral load assay by 8 weeks postinfection
(p.i.), in contrast to high chronic viral replication in SIVmac239
infection. Furthermore, an almost sterilizing immunity against
SIVmac239 was induced in ?5G-infected animals (36). Inter-
estingly, another quintuple-deglycosylation-mutation strain
with mutations at amino acid residues 146, 156, 184, 244, and
247 in Env was created (44) and was demonstrated to share
common features with ?5G in viral replication in animals and
in functions as an attenuated vaccine (20). Since these two
viruses share only one deglycosylation mutation and other mu-
tations distributed differently in surface envelope protein
gp120 (SU), these two studies suggest that heavily glycosylated
Env determines the pathogenicity of HIV/SIV.
To dissect the mechanism for notable containment of ?5G
infection after primary infection, we hypothesized that the Env
of ?5G, a viral protein that differs from that in SIVmac239,
might elicit protective immunity against SIVmac239, because
deglycosylation in Env might alter antigenic properties such as
B-cell and T-cell epitopes and enhance the protective immu-
nity against SIVmac239. For this purpose, we immunized an-
imals with Env of ?5G (?5G Env) or Env of SIVmac239 (the
wild type; wt Env), and examined the effect of these vaccina-
tions against SIVmac239 infection.
MATERIALS AND METHODS
Generation of SU DNA vaccines. DNA vaccine plasmids expressing SIV
mac239 SU or ?5G SU, pJWSUmac239 and pJWSUmac?5G, were constructed
using the expression vector pJW4303 (45). To produce secreted SU efficiently,
the native signal sequence in the SIVmac239 SU gene was replaced with the
human tissue plasminogen activator signal in plasmid pJW4303, and a termina-
tion codon was created at the cleavage site for SU transmembrane (TM) protein
(9). An SIVmac239 SU or ?5G SU DNA sequence was amplified with a pair of
primers, SUmacA (5?-TGTGCTAGCTATGTCACAGTCTTTTATGGTGTAC-
3?) and SUmacB (5?-CCAGGATCCTATTACCTCTTCACATCTGTGGGGG
C-3?). The SUmacA primer consisted of nucleotides (nt) 6923 to 6955 of the
SIVmac239 sequence (GenBank accession number M33262) and the boldface
nucleotides, which were changed to create a NheI site; primer SUmacB consisted
of nt 8412 to 8381 and the boldface nucleotides, which were changed to create a
BamHI site, and the underlined nucleotides, which generated tandem termina-
tion codons. The PCR-amplified fragments were digested with NheI and BamHI
and cloned into the NheI- and BamHI-digested eukaryotic expression vector
pJW4303 to yield pJWSUmac239 and pJWSUmac?5G. These plasmids were
prepared using a Plasmid Mega kit (QIAGEN, Tokyo, Japan).
Generation of Env vaccinia vaccines. Recombinant vaccinia viruses expressing
Env of SIVmac239 or ?5G, WRvvmac239 or WRvv?5G, respectively, were
constructed using a vaccinia virus WR strain (WRvv) as described previously
(15). To excise the entire coding region of the env gene from the cloned SIV
plasmid, BamHI and SmaI sites were introduced by in vitro mutagenesis at 5?-
and 3?-end-flanking sites of the env gene, respectively. Primer B-6808 (5?-GAA
AGAGAAGAAGGATCCCGAAAAAGG-3?) consisted of nt 6796 to 9822 and
the underlined mutations of the BamHI site; S-9537 (5?-TATGAATACTCCC
GGGAGAAACCC-3?) consisted of nt 9527 to 9550 and the underlined muta-
tions of the SmaI site. DNA fragments containing the env gene of SIVmac239 or
?5G were isolated by digesting the mutated plasmids with BamHI and SmaI and
were cloned into the SmaI- and BamHI-digested vaccinia virus vector plasmid
pNZ68K2. To transfer the env gene from a recombinant plasmid to WRvv, the
standard homologous recombination method using CV-1 cells was performed.
Env expression in the recombinant vaccinia virus was confirmed by immunopre-
cipitation. The function of Env was confirmed by CD4- and CCR5-dependent
fusion activity. The recombinant Env-expressing vaccinia viruses obtained were
propagated and titrated in CV-1cells. The two recombinant viruses were prop-
agated with similar kinetics in CV-1 cells.
Expression of SU-expressing plasmids and Env-expressing vaccinia virus in
vitro. CV-1 cells were transfected with equal amounts of the following SU-
expressing plasmids: pJWSUmac239, pJWSUmac?5G, or the vector pJW4303.
Secreted SU metabolically labeled with35S protein labeling mix (PerkinElmer,
Boston, MA) in culture supernatant was concentrated, immunoprecipitated with
plasma from SIVmac239-infected monkeys, and then analyzed by sodium dode-
cyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) as described previously
(40). To examine Env-expressing vaccinia viruses, CV-1 cells were infected with
WRvvmac239, WRvv?5G, or WRvv at a multiplicity of infection of 10, meta-
bolically labeled with35S protein labeling mix overnight, lysed, immunoprecipi-
tated with plasma from SIVmac239-infected monkeys, and then analyzed by
SDS-PAGE as described for the expression of SU-expressing plasmids.
Animals, immunization, and challenge. Twelve juvenile rhesus macaques from
Myanmar or Laos that were seronegative for SIV, simian T-cell lymphotropic
virus, B virus, and type D retroviruses were used. As the polymorphism of major
histocompatibility complex (MHC) genes influenced cellular immune responses
against SIV/HIV infection, MHC II haplotypes and alleles of the macaques were
determined (data not shown). All animals were housed in individual cages and
maintained according to the rules and guidelines for experimental animal welfare
stated by the National Institute of Infectious Diseases. As shown in Fig. 1, the 12
animals were divided into three immunization groups of four animals each: the
SIVmac239 (wt)-Env immunization group (Mm0005, Mm0007, Mm0010,
Mm0012), the ?5G Env immunization group (Mm0001, Mm0002, Mm0003,
Mm0009), and the vector control immunization group (Mm0004, Mm0006,
Mm0008, Mm0011). All animals were inoculated with 1 mg of plasmid DNA in
1 ml of saline, one into each quadriceps femoris at 0, 4, and 8 weeks after the
initial prime immunization (weeks p.p.). The boost consisted of 5 ? 107PFU of
vaccinia virus in 1 ml of phosphate-buffered saline (PBS), administered in two
0.1-ml intradermal inoculations, one into the skin of each femur, and two 0.4-ml
inoculations, one into each quadriceps femoris at 21 weeks p.p. All animals were
VOL. 79, 2005GLYCOSYLATION OF Env IN AIDS VACCINE10387
challenged with 10 50% tissue culture infective doses (TCID50) of SIVmac239
intravenously at 28 weeks p.p.
Viral load measurement. To monitor SIV infection, the plasma viral load was
measured by the real-time-PCR method described previously (36). Viral RNA
was isolated from plasma from the infected animals using a commercial viral-
RNA isolation kit (PE Applied Biosystems, Urayasu, Japan). SIV gag RNA was
amplified and quantified using a commercial RNA reverse transcription (RT)-
PCR kit (TaqMan EZ RT-PCR; PE Applied Biosystems) with the two gag
primers, namely, the forward primer 1224F (5?-AATGCAGAGCCCCAAGAA
GAC-3?), the reverse primer 1326R (5?-GGACCAAGGCCTAAAAAACCC-
3?), and TaqMan probe 1272T (6-carboxyfluorescein-5?-ACCATGTTATGGCC
AAATGCCCAGAC-3?-6-carboxymethylrhodamine). Purified viral RNA (10 ?l)
was reverse transcribed and amplified in a MicroAmp optical 96-well reaction
plate (PE Applied Biosystems) according to the manufacturer’s instructions and
with the following thermal cycle conditions: 1 cycle of three sequential incuba-
tions (50°C for 2 min, 60°C for 30 min, and 95°C for 5 min) and then 50 cycles
of amplification (95°C for 5 s, 62°C for 30 s) in a 7000 Prism sequence detection
system (PE Applied Biosystems). In vitro RNA transcripts were quantified by
optical density at 260 nm (OD260) measurement and branched DNA assay for
SIV viral RNA (Bayer Diagnostics, Tarrytown, N.Y.). RNA equivalent to 10 to
107copies per reaction was used as the standard for each assay. The detection
sensitivity of plasma viral RNA using this method was 1,000 copies/ml.
Flow cytometry. CD4 depletion was monitored by measuring the percentage of
CD4?T cells, memory cells (CD29 high CD4?) T cells (48) in PBMCs. PBMC
samples were purified from a citrate anticoagulant containing blood using stan-
dard Ficoll-Hypaque gradient centrifugation. For flow cytometry, 2 ? 105
PBMCs were reacted with fluorescein isothiocyanate or phycoerythrin-labeled
antibodies (anti-human CD4, Nu-Th/I [Nichirei, Tokyo, Japan]; anti-human
CD8, Leu2a [Becton Dickinson, San Jose, CA]; anti-human CD29, 4B4 [Coulter,
Miami, FL]; anti-monkey CD3, FN-18 [Biosource, Camarillo, CA]; and anti-
human CD20, Leu16 [Becton Dickinson, San Jose, CA]) as previously described
(36, 37, 48).
Peptides. Overlapping peptides were synthesized by Emory University, Micro-
chemical Facility, Winship Cancer Center (Atlanta, GA.). All SIVmac239 viral
proteins except Env, Gag, Pol, Vif, Vpr, Vpx, Tat, Rev, and Nef were covered by
consecutive 20-mer peptides overlapped by 12 amino acids. Env of SIVmac239
was covered by 72 consecutive 25-mer peptides overlapped by 13 amino acids.
Peptides were dissolved in PBS with 10% dimethyl sulfoxide (Sigma Chemical,
St. Louis, Mo.).
rSeV. Recombinant Sendai viruses (rSeV) expressing SIVmac239 Gag, SU, or
?5G SU were used to infect herpesvirus papio-transformed B-lymphoblastoid
cell lines (B-LCLs) to prepare autologous B-LCLs presenting these viral anti-
gens. rSeV Gag expressing unprocessed SIVmac239 Gag and p55 (22, 23) and
rSeV SU and rSeV/?5G SU expressing wt SU and ?5G SU were constructed as
described previously (52) and were also used to infect autologous B-LCLs.
Anti-SIV ELISA. A 1:100 dilution of each plasma sample in PBS (pH 7.4)
containing a blocking reagent (Dainippon Seiyaku, Osaka, Japan) was assayed
for SIV-specific antibody by using a standard enzyme-linked immunosorbent
assay (ELISA) technique with 96-well plates precoated with SIVmac239 virion
lysate. The OD492was measured using a microplate reader (range of absorbance
with linearity, 0 to 3.0; Tecan Japan, Tokyo, Japan) and utilized as a relative
measurement of the antibody titer.
ELISPOT assay. Virus-specific CD4?T cells and CD8?T cells in PBMCs
were measured using a monkey ?-IFN ELISPOT assay kit (U-CyTech, Utrecht,
Cryopreserved PBMCs were thawed and cultured overnight in R-10 medium
(RPMI 1640 [Sigma] supplemented with 10% heat-inactivated, defined fetal
bovine serum [HyClone, Logan, Utah], 55 ?M 2-mercaptoethanol, 50 U/ml
penicillin, and 50 ?g/ml streptomycin). PBMCs were subjected to the depletion
of CD4?cells with magnet beads coated with anti-human CD4 Ab (Dynal ASA,
Oslo, Norway) or subjected to the depletion of CD8?cells with magnet beads
coated with anti-human CD8 Ab (Miltenyi Biotec, Bergisch Gladbach, Ger-
many). Depletion of CD4?or CD8?cells from PBMCs was confirmed by flow
cytometry. Using this depletion method, more than 95% of CD4?or CD8 cells
were removed from PBMCs. These PBMCs were used for ELISPOT assay for
virus-specific CD8?T cells and virus-specific CD4?T cells. Virus-specific stim-
ulation of T cells was performed with autologous B-LCLs pulsed with pooled
peptides for Pol, Vif, Vpx, Vpr, Tat, Rev, and Nef or B-LCLs infected with an
rSeV for Gag, wt Env, and ?5G Env. B-LCLs were incubated with pooled
peptides corresponding to each viral protein at a final concentration of 2 ?g/ml
or infected with rSeV at a multiplicity of infection of 10 at 37°C overnight.
Peptide-pulsed or infected B-LCLs were inactivated with long-wave UV irradi-
ation (19) in the presence of 10 ?g/ml psoralen (Sigma) for 10 min at a distance
of 3.5 cm from a UV light, washed three times with R-10, and then used as
stimulators in an ELISPOT assay. CD4?or CD8?cell-depleted PBMCs were
cultured with these stimulators in an anti-?-IFN Ab-coated ELISPOT plate
(U-CyTech) overnight according to the protocol for the kit. Spots on the
ELISPOT plate were imaged using an Olympus model SZX12 microscope
FIG. 1. Outline of immunization, challenge infection, and blood sampling. Twelve juvenile rhesus macaques were divided into three immu-
nization groups of four animals each: the wt-Env immunization group (Mm0005, Mm0007, Mm0010, and Mm0012), the ?5G Env immunization
group (Mm0001, Mm0002, Mm0003, and Mm0009), and the vector control immunization group (Mm0004, Mm0006, Mm0008, and Mm0011).
Animals were inoculated with a DNA vaccine (pJWSUmac239 for the wt-Env vaccine group, pJWSU?5G for the ?5G Env vaccine group, and
pJW4303 for the vector control group) at 0, 4, and 8 weeks p.p. The boost vaccine consisted of vaccinia virus (WRvvENVmac239 for the wt-Env
vaccine group, WRvvENV?5G for the ?5G Env vaccine group, and the WR strain for the vector control group) administered at 21 weeks p.p. All
animals were challenged with 10 TCID50of SIVmac239 intravenously at 28 weeks p.p. w, weeks; d, day.
10388MORI ET AL. J. VIROL.
(Olympus, Tokyo, Japan) equipped with a digital camera, PDMCIe/OL (Po-
laroid, Cambridge, MA), and analyzed using a personal computer with MAC
SCOPE version 2.61 (Mitani Corporation, Toyama, Japan). The results were
calculated as numbers of spot-forming cells (SFC) per million PBMCs after
subtraction of the background.
Neutralization assay. The original protocol of this neutralization assay was
reported by Means et al. (29). Plasma that was heat inactivated at 56°C for 30 min
was serially diluted and incubated with a fixed concentration of SIVmac239,
?5G, or a macrophage-tropic SIV, 239/envMERT, at room temperature for 1 h.
CEMx174/SIVLTR-SEAP cells were added to the mixture and then incubated at
37°C for 3 days. Secreted alkaline phosphatase activity in the culture supernatant
was measured using a Phospha-Light System (Applied Biosystems). Chemilumi-
nescence was detected with a Wallac Microbeta plate reader.
Statistical analysis. Statistical analysis was based on the Mann-Whitney test
and performed using GraphPad Prism 4.0 software.
Experimental design. We adopted a DNA prime-vaccinia
virus boost regimen to immunize rhesus macaques with wt Env
or ?5G Env as shown in Fig. 1. Twelve macaques were immu-
nized at 0, 4, and 8 weeks after the initial prime immunization
(weeks p.p.) with one of three different DNA expression plas-
mids (n ? 4): pJWSUmac239 expressing SU of SIVmac239,
pJWSU?5G expressing SU of ?5G, or the vector pJW4303. At
21 weeks p.p., all animals were boosted with recombinant WR
vaccinia viruses expressing the respective Env proteins: vac-
cinia virus expressing Env of SIVmac239, vaccinia virus ex-
pressing Env of ?5G, or vaccinia virus (Fig. 1).
Expression of SU DNA plasmids and Env vaccinia viruses in
vitro and in animals. Although ?5G replicated similarly to
wild-type SIVmac239 in animals (36), quintuple deglycosyla-
tion might affect the expression of SU in a plasmid vector and
the expression of Env in the vaccinia virus vector. Thus, we
examined the expression of these vaccines in CV-1 cells. SU
expressions in the wild-type plasmid (pJWSUmac239) and in
the deglycosylated SU plasmid (pJWSUmac?5G) were at sim-
ilar levels (Fig. 2A). The expression and processing of Env in
the wild type (WRvvENVmac239) and in the deglycosylated
Env mutant vaccinia virus (WRvvENV?5G) were also at sim-
ilar levels (Fig. 2B). The reduced molecular size of the proteins
due to deglycosylation was confirmed by PAGE (Fig. 2). As the
amount of secreted SU in the supernatant by DNA transfec-
tion was comparable to that of Env in the cell lysate from CV-1
cells infected with WRvvEnv, a high expression of SU was
achieved in a rev-independent manner by the pJW403 expres-
sion plasmid as described previously (9).
The expression of Env vaccines in the immunized animals
was indirectly estimated by Env-specific antibody responses
measured by a peptide ELISA using overlapping Env peptides.
Env peptide-specific Ab was detected from 11 weeks p.p. after
immunization with DNA vaccines, whereas there was no sig-
nificant difference in the titers and the specificity of the re-
sponses between the two vaccine groups (data not shown),
suggesting similar amounts of Env expressed in animals immu-
nized with either Env vaccine. To examine the protective effect
of the Env vaccines, all animals were challenged with 10
TCID50of SIVmac239 intravenously at 28 weeks p.p.
Cellular immune responses elicited by Env vaccines. The
DNA prime-vaccinia virus boost regimen has been used in
many studies, has successfully induced a high frequency of
virus-specific CD8?T cells in macaques, and has conferred
protective immunity against chimeric simian/human immuno-
deficiency virus (SHIV) (3, 27, 45). We therefore examined the
vaccine-induced Env-specific T-cell responses by IFN-? ELIS-
POT assay. Since deglycosylation in Env might change T-cell
epitopes in SIVmac239, we measured the wt-SU and ?5G
SU-specific T-cell response by using autologous B-LCLs in-
fected with recombinant Sendai viruses expressing either wt
SU and/or ?5G SU, respectively.
Although there was a tendency for more ELISPOT-positive
cells to be observed by homologous SU than heterologous SU,
comparable results were obtained by both assays (Fig. 3A and
B). As vaccinated animals were challenged with SIVmac239,
the results from the wt-SU assay were subsequently used to
assess the SU-specific immune response. Immunization with
the DNA vaccine induced only marginal SU-specific CD8?T
cells or CD4?T cells at 11 weeks p.p.; however, boost immu-
nization with recombinant WR vaccinia virus significantly in-
creased SU-specific CD8?T cells and CD4?T cells in PBMCs
at 26 weeks p.p. (Fig. 3A, B, and C). Notably, SIVmac239 Env
(wt Env) induced twofold more SU-specific CD8 T cells (mean,
770 SFC per million PBMCs; range, 540 to 880) responding to
wt SU than ?5G Env (mean, 320; range, 110 to 400) (P ?
0.029) (Fig. 3A and C). Similarly, twofold more SU-specific
CD4?T cells were observed in wt-Env vaccinees (mean, 1,260;
range, 840 to 1,710) than in ?5G Env vaccinees (mean, 680;
range, 150 to 1,260) at 26 weeks p.p. (P ? 0.11) (Fig. 3B and
C). Thus, a twofold-greater number of both SU-specific CD4?
T cells and CD8?T cells were induced in SIVmac239 Env
vaccinees than in ?5G Env vaccinees at 26 weeks p.p. In vector
controls, only negligible SU-specific CD4?T cells and CD8?T
cells were detected in PBMCs at 26 weeks p.p. (Fig. 3A and B).
Humoral immune response elicited with Env vaccines. The
anti-Env Ab titer was examined by SIVmac239 virion lysate
ELISA. Anti-SIV Ab was detected in both wt-Env vaccinees
and ?5G Env vaccinees after an rVV boost (Fig. 4) (26 weeks
p.p.). Anti-SIV Ab titers were comparable between the two
Next, we examined the NAb against either SIVmac239,
?5G, or a macrophage-tropic mutant, 239env/MERT (33, 35),
in the two vaccine groups. Macrophage-tropic SIVs were
highly susceptible to neutralization by plasma from most SIV-
infected macaques (29), whereas SIVmac239 was highly resis-
tant to neutralization as were most clinical isolates of HIV-1
FIG. 2. Expression of SU and Env by SU-expressing DNA vaccines
and Env-expressing vaccinia viruses. A: SU secreted in supernatant
from CV-1 cells transfected with SU-expressing plasmids. Lane 1,
pJW4303 vector; lane 2, pJWSUmac239; lane 3, pJWSUmac?5G. B:
Env in cell lysates of CV-1 cells infected with recombinant vaccinia
viruses. Lane 1, WRvv; lane 2, WRvvmac239; lane 3, WRvv?5G.
VOL. 79, 2005GLYCOSYLATION OF Env IN AIDS VACCINE 10389
(21, 29, 30). Plasma at 26 weeks p.p. from all immunized
animals failed to neutralize not only SIVmac239 but also a
multiple-deglycosylation-mutation strain, ?5G (Table 1); in
contrast, these plasma specimens did neutralize 239env/
MERT. Furthermore, a marked difference was observed be-
tween the two vaccine groups. The NAb titer in the wt-Env
vaccine group was eightfold higher than in the ?5G Env vac-
cine group (Table 1). The difference of this immune response
between the two vaccine groups was significant (P ? 0.029).
SIV replication in Env-immunized animals. As described
above, wt-Env vaccine and ?5G Env vaccine induced different
magnitudes of virus-specific cellular and humoral immunity in
macaques. To examine the effect of the two vaccines, we chal-
lenged the vaccinated animals with SIVmac239. Viral loads in
vector controls were mostly consistent with our previous results
with SIVmac239-infected rhesus macaques (36, 48). The mean
peak viral load at 2 weeks p.i. was 1.4 ? 107copies/ml, with a
range of 0.5 ? 107to 2.2 ? 107copies/ml. Viral loads in chronic
infection diverged into two patterns (Fig. 5A). Subsequent to
the set point at 20 weeks p.i., the viral loads in three animals
increased more than 104copies/ml. In contrast, viral loads in
one animal (Mm0011) remained as low as 1,000 copies/ml up
to 45 weeks p.i.
Compared with the vector controls, viral loads in wt-Env
FIG. 3. Env-specific CD4?T-cell and CD8?T-cell responses in 12 macaques. A: Env-specific CD8?T cells in PBMCs were measured by
ELISPOT assay for IFN-? in three groups. B: Env-specific CD4?T cells in PBMCs were measured by ELISPOT assay for IFN-? in three groups.
ELISPOT results are colored as follows: ?5G SU-specific T cells (red), wt-SU-specific T cells (green), and TM-specific T cells (yellow). Arrows
with a dotted line, arrows with broken line, and arrows with a solid line indicate the time of the third DNA vaccination at 8 weeks p.p., the time
of the vaccine boost at 21 weeks p.p., and the time of SIVmac239 challenge at 28 weeks p.p., respectively. C: Comparison of SU-specific CD8?T cells
and CD4?T cells in PBMCs among the wt-Env vaccine group, the ?5G Env vaccine group, and the vector control group at 26 weeks p.p. and 4, 7, and
12 days p.i. The numbers of SFC responding to SIVmac239 SU were used to compare the effects of the two vaccines. w, weeks; d, days.
10390MORI ET AL.J. VIROL.
vaccinees were markedly reduced (Fig. 5B). Peak viral loads at
2 weeks p.i. (mean, 1 ? 106copies/ml; range, 0.8 ? 106to 1.2
? 106copies/ml) were 1-log lower than those in the vector
controls. Furthermore, viral loads decreased to as low as 1,000
copies/ml by 8 to 20 weeks p.i., remaining low until autopsy at
45 weeks p.i.
Unexpectedly, viral loads in the ?5G Env vaccine group
resembled those in vector controls (Fig. 5C). Peak viral loads
(mean, 2.4 ? 106copies/ml; range, 0.9? 106to 4.2 ? 106
copies/ml) were slightly lower than those in vector controls. Set
points and viral loads in the chronic phase were similar to
those of vector controls.
In summary, as shown by the mean viral loads in primary and
chronic infection (Fig. 5D) and statistical analysis (Fig. 5E),
the effects of vaccination differed between the wt-Env vaccine
and ?5G Env vaccine. In the effect on primary infection (up to
6 weeks p.i.), wt-Env vaccination decreased viral loads more
extensively and significantly than ?5G Env vaccination (P ?
0.029 versus P ? 0.057); however, in chronic infection (viral
loads after 8 weeks p.i.), significant reductions in viral loads
compared with those in vector controls were seen only in the
wt-Env vaccine group and not the ?5G Env vaccine group (Fig.
5E). Collectively, wt-Env vaccination induced significantly ef-
fective immunity to control SIVmac239 infection, whereas
?5G Env vaccination induced a marginal effect seen only in
primary and not in chronic infection.
CD4?T-cell subsets in PBMCs. CD4 cell depletion is a
primary manifestation indicating immune disorder in HIV/SIV
infection. As CD4 depletion results from HIV/SIV infection in
lymphatic tissue, it correlates with the extent of viral replica-
tion. Accordingly, viral loads were correlated mostly with CD4
depletion (Fig. 5 and 6A). Despite fluctuations due to immu-
nizations and the challenge infection, the percentage of CD4?
T cells in wt-Env-immunized animals in the chronic phase
recovered to the levels at the initiation of the experiment. By
contrast, in vector controls and ?5G Env vaccinees, the per-
centage of CD4?T cells decreased in the chronic phase.
Among them, an extensive decrease in CD4?T cells occurred
in animals with high viral loads in the chronic phase (Mm0001,
Mm0008, and Mm0009) (Fig. 5 and 6A). However, in the other
animals, the levels of CD4?T cells remained as before the
challenge (Mm0003, Mm0011).
A subset of CD4?CD29 high cells, approximately corre-
sponding to memory CD4?T cells, is useful for diagnosing a
deterioration in the immune function in animals with AIDS
(26, 38, 48). Although this parameter usually correlates with
the percentage of CD4?T cells, remarkable differences were
noted between two Env vaccine groups after the challenge
infection. First, all animals in the wt-Env vaccine group showed
an increased percentage of this subset in the chronic phase
(Fig. 6B). Second, three of the ?5G Env vaccinees had a
marked decrease after the challenge infection (Mm0001,
Mm0002 and Mm0009), whereas the remaining animal
(Mm0003) showed an increased percentage of this subset. In
FIG. 4. Humoral immune response during immunization and after challenge infection. The OD492was used as a relative measurement of
anti-SIV ELISA antibody titer.
TABLE 1. Neutralizing-antibody titers in the vaccinated macaques
at 26 weeks p.p.
aReciprocal of the dilution of plasma giving 50% inhibition of SIV replication.
bThe difference in NAb levels between the two vaccine groups was significant
(P ? 0.0029).
VOL. 79, 2005GLYCOSYLATION OF Env IN AIDS VACCINE 10391
vector controls, this subset remained in the range before the
challenge infection in all animals but one (Fig. 6B).
Env-specific-T-cell immunity after the challenge infection.
The magnitude of Env-specific T cells after the challenge in-
fection is assumed to be influenced not only by vaccination but
also by viral replication. Namely, SU-specific T cells at 4 days
p.i. and those at 12 days p.i. were likely influenced by the
former and the latter respectively. The magnitudes of SU-
specific CD4?T cells and CD8?T cells at 4 days p.i. were
comparable to those before challenge at 26 weeks p.p. (Fig. 3A
and B); therefore, twofold-more SU-specific CD8?T cells and
CD4?T cells were present in wt-Env vaccinees than in ?5G
Env vaccinees up to 4 days p.i. (Fig. 3C). However, this differ-
ence in the magnitudes of SU-specific CD8?T and CD4?T
cells was not sustained at 7 and 12 days p.i. (Fig. 3C). Present
with robust viral replication in primary infection, SU-specific
CD4?T cells immediately decreased to an undetectable level
at 12 days p.i. In contrast, SU-specific CD8?T cells increased
(Fig. 3A and B). Subsequently, SU-specific CD8?T cells grad-
ually decreased to very low or undetectable levels by 34 weeks
p.i. (Fig. 3A). Thus, vaccine-induced SU-specific CD8?T and
CD4?T cells were sustained only for a short period of time
after challenge infection in both Env vaccine groups.
SIV-specific T-cell immunity after challenge infection. De-
spite an Env vaccination, robust SIV infection occurred shortly
after the challenge infection (Fig. 5B and C). Consequently,
SIV-specific CD8?T cells and CD4?T cells were elicited not
only in vector controls but also in Env vaccine groups (Fig. 7A
and B). To examine the effect of these SIV-specific T cells on
the control of SIV infection, all animals were divided into SIV
infection-controlled (controlled) and SIV infection-uncon-
trolled (uncontrolled) animals. Viral loads in chronic infection
and the percentage of CD4?cells in PBMCs were used to
classify the animals as controlled or uncontrolled (Fig. 6A). All
animals in the wt-Env vaccine group, Mm00011 in vector con-
trols, and Mm0003 in the ?5G Env vaccine group were
grouped as control animals. The remaining animals, Mm0004,
Mm0006, and Mm0008 in vector controls and Mm0001,
Mm0002, and Mm0009 in the ?5G Env vaccine group were
grouped as uncontrolled animals. Notably, SIV-specific CD4?
T cells as well as the percentage of CD4?CD29H cells re-
mained high in the chronic phase in controlled animals (Fig.
7B and 6B, respectively).
Although overall SIV-specific CD8?T cells were high in
Env-vaccinated controlled animals, such correlation was not
seen in vector controls grouped as uncontrolled animals (Fig.
7A). Therefore, to examine the relevance of virus-specific T
cells to the control of SIV infection, the magnitudes of every
viral-protein-specific T cell in controlled and uncontrolled an-
imals were compared. As shown in Fig. 7C, Gag-specific CD8?
T cells and CD4?T cells, and Tat/Rev-specific CD4?T cells
FIG. 5. Plasma viral loads after SIVmac239 challenge infection. Plasma viral load was measured by real-time PCR with a detection limit of
1,000 copies/ml. A: wt-Env vaccine group; B: ?5G Env vaccine group; C: vector controls; D: comparison of viral loads among three groups; E:
comparison of viral loads during the primary infection (5 days to 6 weeks p.i.) and chronic infection (8 weeks to 45 weeks p.i.) among three groups.
Viral load was determined by averaging over a period of time.
10392 MORI ET AL.J. VIROL.
were induced, with statistical significance (P ? 0.05), in the
The heavily glycosylated structure of Env has been consid-
ered a main cause of chronically persistent viral replication and
the pathogenicity of HIV/SIV, primarily because it potentially
interferes with the development of the host immune response
associated with protective immune functions, such as NAb and
CTL (10, 36, 44). This characteristic constitutes the primary
reason for the difficulty of developing effective vaccines. We
therefore examined the efficacy of a deglycosylated-Env vac-
cine and compared it with the wt-Env vaccine. This study
showed that quintuple deglycosylation neither improved the
immunogenicity of the wt-Env vaccine nor elicited NAb
against SIVmac239. This was in contrast to what occurred with
?5G infection in rhesus macaques, because the host response
elicited by ?5G infection not only contained ?5G infection but
also protected the animals from SIVmac239 challenge infec-
tion (36). This study therefore suggested that an almost ster-
ilizing immunity against SIVmac239 induced in ?5G-infected
animals could not be explained by the immunogenicity of ?5G
Env; instead, it is likely associated with the property of ?5G as
an attenuated virus. In fact, ?5G was more neutralization-
sensitive than SIVmac239 (36). Alternatively, the immuno-
genic property of Env in ?5G could not successfully be dupli-
cated by immunization with a ?5G Env DNA prime-vaccinia
virus boost regimen. Therefore, another immunization regi-
men might be able to elicit the protective immune response
induced by ?5G infection.
The Env vaccine is superior to other vaccines containing
other viral proteins with respect to the induction of NAb;
however, both the ?5G Env vaccine and the wt-Env vaccine
could not induce detectable NAb against either SIVmac239 or
?5G. Instead, the wt-Env vaccine induced higher NAb against
macrophage-tropic SIV than the ?5G Env vaccine. Notably,
this parameter most significantly correlated with the efficacies
of the two Env vaccines. As Ab neutralized the macrophage-
tropic variant 239/envMERT, which has only four separate
amino acid substitutions distributed in env of SVmac239 (34),
it might recognize unknown epitopes conserved between
SIVmac239 and 239/envMERT. On the other hand, ?5G Env
may not sufficiently present this epitope due to mutations.
Regarding the role of nonneutralizing Ab for the control of
SIVmac239 infection, it is assumed that, as the neutralization
assay did not necessarily reflect in vivo conditions, such non-
neutralizing Ab with potential virus-binding ability may inter-
fere with SIVmac239 infection in animals. Alternatively, Ab
FIG. 6. CD4?T cells in PBMCs from rhesus macaques during immunization and after the challenge infection. A: Percentage of CD4?T cells
in PBMCs; B: percentage of CD4?CD29high T cells in PBMCs.
VOL. 79, 2005GLYCOSYLATION OF Env IN AIDS VACCINE 10393
might play a role in other effector functions, such as antibody-
dependent cell-mediated cytotoxicity to eliminate the infected
cells. The antibody-mediated enhancement of viral antigen
processing and cross presentation is also a mechanism poten-
tially related to the control of SIV infection in vivo (49).
Reduced immunogenicity in the ?5G Env vaccine was also
noted in cellular immunity. The levels of stimulation of anti-
gen-specific CD8?T cells and CD4?T cells are MHC I and
MHC II dependent, respectively. As the macaques in this study
have different MHC haplotypes (data not shown), the magni-
tude and breadth of SIV-specific T cells should vary among the
animals. Nevertheless, the magnitude of SU-specific CD8?T
cells and CD4?T cells in PBMCs was greater in the wt-Env
vaccine group than in the ?5G Env vaccine group. Although
the expression of SU by expressing plasmids and that of Env by
the vaccinia virus vector elicited by either the wt-Env vaccine
or ?5G Env vaccine were indistinguishable in cultured cells
(Fig. 2), wt-Env might persist longer than ?5G Env in vacci-
nated animals. T-cell epitopes in the wt-Env vaccine might
therefore be more efficiently presented on MHC molecules in
antigen-presenting cells than in the ?5G Env vaccine. Differ-
ences in glycosylation levels might also affect some processes in
antigen-presenting cells associated with the presentation of
T-cell epitopes in Env.
Taking all results together, Env glycosylation might affect
the presentation of B-cell epitopes and T-cell epitopes re-
quired for Ab-mediated and T-cell-mediated immunities re-
lated to the control of SIV infection.
FIG. 7. SIV-specific CD8?T-cell and CD4?T-cell responses in 12 animals. A: SIV viral-protein-specific CD8?T cells in PBMCs were
measured by ELISPOT assay for IFN-? in three groups: vector controls, wt-Env vaccine group, and ?5G Env vaccines. B: SIV viral-protein-specific
CD4?T cells in PBMCs were measured by ELISPOT assay for IFN-? in three groups. ELISPOT results of individual SIV proteins are colored
as follows: Gag (red), Nef (green), Tat/Rev (blue), Vif/Vpr/Vpx (yellow), and Pol (pink). C: Comparison of cumulated CD8?T cells or CD4?T
cells specific to the viral proteins Gag, Pol, Nef, Tat/Rev, and Vif/Vpr/VpX between SIV infection-controlled and uncontrolled animals. w, weeks;
10394MORI ET AL. J. VIROL.
As seen in viral loads and SU-specific T cell levels after
challenge infection (Fig. 3 and 5), the effect of vaccination was
limited. That seemed related to the development of escape
mutants. Therefore, distinctive cellular immune responses af-
ter the challenge infection were also implicated in the control
of SIVmac239 replication. The magnitude of virus-specific
CD8?T cells did not always correlate with the suppression of
viral replication as reported previously (1, 6), particularly in
vector controls (Fig. 5 and 7A); however, selected epitope-
specific CTL responses might be associated with infection con-
trol. Gag-specific CTLs are such candidates, because a high
magnitude of Gag-specific CD8?T cells was significantly elic-
ited in five control animals (Fig. 7C). The magnitude of Gag-
or Tat/Rev-specific CD4?T cells was statistically correlated
with infection control (Fig. 7C). This may simply indicate a
lower depletion of virus-specific CD4?T cells in animals with
lower viral loads as reported previously (11). Alternatively,
these virus-specific CD4?T cells may play an important role in
protective immunity (39). Taken together, these results impli-
cated the dominant role of selected epitope-specific CD4?T
cells and CD8?T cells for the control of SIVmac239 infection.
The challenge virus that should be used has been an impor-
tant issue in AIDS vaccine studies (8, 10, 12). Many studies
have reported impressive efficacy in a pathogenic-SHIV ma-
caque model (3, 4, 45, 46); however, pathogenic SHIVs use
CXCR4 as a coreceptor, whereas the majority of clinical iso-
lates of HIV-1 use CCR5 (13, 27). Therefore, the challenge
virus for an AIDS vaccine study should be an R5 virus, such as
SIV (10). Consistent with this concern, a DNA prime–modi-
fied-vaccinia virus Ankara boost regimen, inducing broad SIV-
specific T-cell responses, reduced the initial viral replication
but did not prevent disease progression against SIVmac239
challenge (18). Thus, vaccine studies using pathogenic SHIV
should be reevaluated by using an R5 virus (10).
Matano et al. reported that a DNA prime-Sendai virus boost
regimen induced the CTL-based control of SIVmac239 in rhe-
sus macaques (27). This study demonstrated that a DNA
prime-vaccinia virus WR boost regimen expressing only Env
controlled the chronic infection of SIVmac239 in rhesus ma-
caques. The relatively lower viral loads in macaques from My-
anmar or Laos than in those of Indian origin might contribute
to the control of SIVmac239 infection. Nevertheless, it is im-
portant that these two studies demonstrated the efficacies of
the two vaccine regimens against highly pathogenic SIVmac239.
In earlier studies, other R5 SIVs were used as a challenge virus
for an efficacy study of vaccine candidates. An Env-based vac-
cine in vaccinia virus vector priming and subunit protein boost-
ing protected cynomologous macaques against homologous
SIVmne clone E11S (42). In recombinant modified vaccinia
virus, Ankara viruses expressing Gag-Pol and/or Env exhibited
vaccine efficacy because of reduced viremia and the increased
SIVsmE660 (41). Accordingly, the efficacy of vaccine candi-
dates might be influenced by the experimental conditions.
Thus, well-defined animal models with detailed virological,
immunological, and genetic information and suitable challenge
viruses are required for the evaluation of vaccine candidates
and the development of an AIDS vaccine.
This study demonstrated the importance of Env as a com-
ponent of the AIDS vaccine, and Env-specific CD8?and
CD4?T cells and nonneutralizing Env-specific Ab were sug-
gested as protective immunity components. Quintuple degly-
cosylation in Env reduced vaccine efficacy and Env-specific
immune responses. Env may therefore be comprised of appro-
priate antigenic properties to elicit humoral and cellular im-
mune responses required for protective immunity against ho-
mologous or allele-specific target SIV/HIV. These properties
could be modified by the alteration of glycosylation.
In conclusion, although Env is an important immunogen for
the AIDS vaccine, Env properties, including glycosylation,
should be carefully considered to design vaccines specific to the
We thank Kayoko Ueda for excellent technical assistance.
This work was supported by AIDS research grants from the Health
Sciences Research Grants, from the Ministry of Health, Labor, and
Welfare in Japan, and from the Ministry of Education, Culture, Sports,
Science and Technology in Japan.
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