AIDS Research Center, National Institute of
Infectious Diseases, Tokyo 162-8640,1
Tsukuba Primate Center for Medical Sciences, National Institute
of Infectious Diseases, Tsukuba 305-0843,2
Department of Bioregulation, Mie University School of
Medicine, Tsu 514-8507,3 and Department
of Viral Infections, Research Institute for Microbial Diseases,
Osaka University, Osaka 565-0871,5 Japan, and
Institut für Virologie und Immunbiologie,
Universität Würzburg, Würzburg D-97078,
Germany4
We previously generated a mutant of simian immunodeficiency virus
(SIV) lacking 5 of a total of 22 N-glycans in its external envelope
protein gp120 with no impairment in viral replication capability and
infectivity in tissue culture cells. Here, we infected rhesus macaques
with this mutant and found that it also replicated robustly in the
acute phase but was tightly, though not completely, contained in the
chronic phase. Thus, a critical requirement for the N-glycans for the
full extent of chronic infection was demonstrated. No evidence
indicating reversion to a wild type was obtained during the observation
period of more than 40 weeks. Monkeys infected with the mutant were
found to tolerate a challenge infection with wild-type SIV very well.
Analyses of host responses following challenge revealed no neutralizing
antibodies against the challenge virus but strong secondary
responses of cytotoxic T lymphocytes against multiple antigens,
including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation
mutant appeared to represent a novel class of SIV live attenuated vaccine.
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INTRODUCTION |
Apparently potent humoral and
cytotoxic T lymphocyte (CTL) responses are elicited in humans following
infection with human immunodeficiency virus type 1 (HIV-1) (3,
23). However, these responses can only partially control viral
replication, allowing the establishment of a long-term persistent
infection, in which vigorous viral replication and elimination of the
virus by the host responses take place (7). Essentially
the same feature is shared with a simian counterpart, simian
immunodeficiency virus (SIV), and appears to be the background
underlying the difficulty in developing HIV-1 vaccines of sufficient
protective efficacy (9, 18). One possible factor
contributing to viral ability to evade host responses and to cause
persistent infection may be heavy glycosylation of the gp120 external
envelope glycoproteins, because viral surface glycans are thought to
help shield the potential epitopes from immune recognition. In
addition, the glycans tend to protect the proteins from proteolysis,
suggesting that heavily glycosylated proteins are less efficiently
antigen-presented than those with fewer glycans. On the other hand,
enveloped viruses causing acute infection, which is eradicable
naturally or by vaccination, are generally sparsely glycosylated. For
instance, there are 26 and 23 potential N-linked glycosylation sites in
the gp120s of HIV-1 strain SF2 and SIV strain mac239, respectively,
whose polypeptide backbones are 482 and 503 amino acids long,
respectively (11, 16, 17, 22). On the other hand, measles
virus (Edmonston strain), also targeting cells constituting the immune
system but readily eradicable by immune responses, contains only 5 potential N-linked glycosylation sites on its 617-residue-long
receptor-binding protein H (22).
In a series of mutagenesis experiments to silence the potential
N-glycosylation sites of SIVmac239 individually and in combination, we
found that 22 of the 23 potential sites are actually glycosylated and
that 18 are dispensable for viral infectivity, while 4 are essential
(22). Two of the dispensable glycans appeared to be a
strong downmodulater of viral replication ability, because removal of
either markedly enhanced viral infectivity and replication. The
remaining 16 were regarded as neutral, because their removal neither
increased nor decreased infectivity (22). There was a
striking position specificity for these three functionally different glycans, because most of the neutral sites were mapped to the N-terminal half of gp120, while the downmodulating sites mapped to the
C terminus and the essential sites mapped to the central portion. In
addition, we were able to remove up to five glycans in combination
(22). Thus, a panel of deglycosylation mutants are now
available to define the role of each glycan as well as glycans in
combination in the context of SIV replication and pathogenesis in vivo
in rhesus monkeys (22). In this study, we have used a
mutant with five glycans removed, here termed 
5G, and show that it
is fully capable of replication in the acute phase but greatly, albeit
not completely, controlled in the subsequent chronic phase in rhesus
macaques. This quintuple deglycosylation mutant was also found to
behave as a live attenuated vaccine in macaques, conferring potent
protective immunity against wild-type SIV.
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MATERIALS AND METHODS |
Viruses.
SIVmac239 and its derivative 5G were used. The
5G variant was created by mutagenesis of the parental infectious DNA
clone so that the asparagine residues for N-glycosylation at positions 79, 146, 171, 460, and 479 were converted to glutamine residues (22). Actual removal of the five glycans was biochemically
confirmed (22). The stocks of SIVmac239 and 5G were
obtained by DNA transfection of the respective infectious DNAs into
COS1 or SW480 cells. For monkey infection studies, these virus stocks
were propagated in phytohemagglutinin-stimulated peripheral blood
mononuclear cells (PBMCs) from rhesus monkeys and their titers (50%
tissue culture-infective doses [TCID50]) were determined
utilizing herpes saimiri virus-transformed cynomolgus CD4+
T cells (CyfT/HSV) (21).
SIV infection of rhesus macaque PBMCs.
PBMCs (5 × 106) were stimulated with 5 ml of 1-µg/ml
phytohemagglutinin in RPMI medium supplemented with 10% fetal calf
serum, penicillin (50 U/ml), and streptomycin (50 µg/ml) (R10 medium) for 2 days. PBMCs were infected with 5G or SIVmac239 virus stock containing 5 ng of p27 Gag antigen prepared by DNA transfection of
SW480 cells. PBMCs were maintained in R10 medium supplemented with
human interleukin-2 (100 U/ml). Culture supernatant collected every 3 to 4 days was subjected to the SIV p27 Gag antigen assay (Coulter,
Tokyo, Japan).
Animal infection studies.
Juvenile rhesus macaques
seronegative for SIV, simian T-lymphotropic virus, B virus, and type D
retroviruses were used. All animals were housed in individual cages and
maintained according to the rules and guidelines for experimental
animal welfare of our institution. Infection was initiated
intravenously with 100 or 500 TCID50 of the viruses.
Determination of plasma viral load by real-time PCR.
Viral
RNA was isolated from plasma with a commercial viral RNA isolation kit
(Boehringer Mannheim, Tokyo, Japan). SIV gag RNA was
amplified and quantified with a commercial RNA reverse transcription-PCR kit (TaqMan EZ RT-PCR; PE Applied Biosystems, Urayasu, Japan) with the gag forward primer 1224F
(5'-AATGCAGAGCCCCAAGAAGAC-3') and reverse primer 1326R
(5'-GGACCAAGGCCTAAAAAACCC-3') and TaqMan probe 1272T
(FAM-5'-ACCATGTTATGGCCAAATGCCCAGAC-3'-TAMRA). Purified viral RNA (10 µl) was reverse transcribed and amplified in a
MicroAmp optical 96-well reaction plate (PE Applied Biosystems)
according to the manufacturer's instructions and the following thermal
cycle conditions: one cycle of three sequential incubations (50°C for 2 min, 60°C for 30 min, and 95°C for 5 min) and 50 cycles of
amplification (95°C for 5 s and 62°C for 30 s) was
performed in a 7700 Prism sequence detection system (PE Applied
Biosystems). In vitro RNA transcripts were quantified by optical
density at 260 nm (OD260) measurement and b-DNA assay for
SIV viral RNA (Bayer Diagnostics, Tarrytown, NY). RNA equivalent to
107 to 10 copies per reaction was used as a standard for
each assay. The detection sensitivity of plasma viral RNA by this
method was 1,000 copies/ml.
Quantification of viral DNA in PBMCs by serial dilution and
nested PCR and their sequencing.
PBMCs were isolated from
citrate-treated blood by standard Ficoll-Hypaque gradient
centrifugation. Cellular DNA was extracted from 106 cells
and dissolved in 200 µl of elution buffer with a commercial DNA
purification kit (Qiagen, Tokyo, Japan). Serial 10-fold diluted DNA (5 or 10 µl) was subjected to nested PCR with the Ex-Taq PCR kit
(Takara, Tokyo, Japan). The first PCR was done with the first primer
pair env-1F (nucleotides 6537 to 6559)
(5'-CACGAAAGAGAAGAAGAACTCCG-3') and env-1R (nucleotides 7324 to 7304) (5'-GCAAAGCATAACCTGGAGGTG-3'). The numbering is for
SIVmac239 (24). The env sequence
encompassing the V1 to V2 region, where two deglycosylation mutations,
at amino acids (aa) 146 and 171, reside, was amplified with the second primer pair, F6875 (nt 6875 to 6863) (5'-GGCAACTCTTTGAGACCTC-3') and B7164 (nt 7164 to 7140)
(5'-CCAAGTTTCATTGTACTCTTTTTTC-3'). This technique detects 1 copy per 50,000 cells. The number of PBMCs was determined with a blood
cell counter (Sysmex, Kobe, Japan). PCR-amplified DNA was detected by
regular agarose gel electrophoresis. The cloned env DNAs
were sequenced to define whether the viral DNA was derived from the
5G virus or the challenge virus SIVmac239.
Determination of viral load in PBMCs by coculture with monkey
CD4+ T cells.
Fourfold serial dilutions of PBMCs
(starting at 106 in 0.5 ml) obtained from the infected
monkeys were added to 2 × 105 CyfT/HSV cells (in 0.5 ml) in duplicate in 24-well plates and cultured for 4 weeks. After 3 to
4 weeks, the wells were scored for the development of cytopathic effect
due to SIV infection. Although CyfT/HSV cells are very sensitive to
cytopathic effect, SIV infection was confirmed by the presence of p27
Gag antigen in culture supernatants with a commercial SIV p27 assay kit
(Coulter, Tokyo, Japan).
Anti-SIV ELISA.
A 1:100 dilution of each plasma sample in
phosphate-buffered saline (pH 7.4) containing a blocking reagent
(Dainippon Seiyaku, Osaka, Japan) was assayed for SIV-specific antibody
using a standard enzyme-linked immunosorbent assay. (ELISA) technique
in 96-well plates coated with SIVmac239 virion lysate. The
OD492 was recorded and used as a relative measure of
antibody titer.
Neutralization assay.
The method originally described by
Means et al. (19) was employed for neutralization.
Heat-inactivated (56°C for 30 min) plasma was serially twofold
diluted and tested for inhibition of SIV infection in CEMx174 cells
harboring the SIV long terminal repeat (LTR)-driven secreted alkaline
phosphatase (SEAP) reporter gene (CEMx174 SEAP cells). Diluted plasma
(25 µl) was incubated with SIV (100 µl) in a 96-well plate at room
temperature for 1 h. CEMx174 SEAP cells (20,000 cells) were added
to the mixture and incubated for 3 days (SIVmac239 infection) or 5 days
(5G infection). SEAP activity in the culture supernatant was assayed using the Tropix Phospha-Light assay kit (PE Applied Biosystems). Chemiluminescence was measured with a Wallac Micro-beta plate reader
(Amersham Pharmacia Biotech, Tokyo, Japan).
CTL assay.
The CTL assay method used was previously
described (29). PBMC samples stored at 
150°C were
thawed and cultured in RPMI medium with concanavalin A (5 µg/ml) at
106 cells per ml for 3 days, washed, and then maintained
for another 3 days in medium supplemented with human interleukin-2 (2 ng/ml). Target cells, autologous herpesvirus papio-transformed B-LCL
cells, were infected with recombinant vaccinia virus (VV) expressing the SIV proteins SIVmac251 Gag-Pol, SIVmac239 Nef, SIVmac239 Env, or
5G Env or the parental VV (NYCBH strain) at 37°C for 16 h. They were labeled with 51Cr and then incubated with
effector cells at 37°C for 5 h. Specific lysis was calculated as
percent SIV-specific lysis minus percent lysis of control VV
(NYCBH)-infected target cells.
| |
RESULTS |
Viral loads following 5G infection.
The mutant 5G lacks
specific sites for N glycosylation at the asparagine residues at amino
acid positions 79, 146, 171, 460, and 479. The first three are neutral,
and the last two downmodulate viral infectivity in cell culture
(22). The 5G mutant replicated as efficiently as
parental SIVmac239 in T-cell lines. Both the mutant and wild-type
viruses were propagated in rhesus monkey PBMCs. They replicated with
similar kinetics and reached comparable peak titers, 45 and 30 ng/ml,
respectively, of p27 antigen on day 21. We then intravenously infected
two rhesus macaques with 500 TCID50 of the 5G virus and
another two with the same dose of the parental SIVmac239 and determined
viral RNA levels in the plasma. SIVmac239 and 5G were both found to
replicate vigorously in the early acute phase, the viremia peaking at 2 weeks postinfection (p.i.) with comparably high titers (Fig.
1). Thus, 5G did not appear to be
attenuated at all, at least in this early stage of infection. At 3 weeks p.i., the viral loads began to decrease sharply in both cases,
but dramatic differences in the set point and subsequent viral load in
the chronic phase were seen. In both monkeys infected with 5G, the
set points were mostly below the level of detection (1,000 copies/ml)
and low loads or loads just above the detection level persisted for up
to 32 weeks p.i. Compared with these, both the set points of wild-type
infection and the chronic viral loads were remarkably higher for
monkeys infected with SIVmac239, except that one of the monkeys
displayed a low viral load at 20 weeks p.i.
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FIG. 1.
Replication of the 5G deglycosylation mutant and the
parent SIVmac239 in rhesus macaques. At various weeks (wks) times after
intravenous injection of 500 TCID50 of each virus, viral
loads in plasma were determined.
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The replication patterns suggested the possibility that 5G totally
lacked the ability to replicate in the chronic phase or was totally
controlled by host immune responses. To address this issue, we
attempted to isolate the virus from PBMCs of monkeys 22 and 23. At 8 weeks p.i., 40,000 and 8,000 PBMCs were required to isolate the virus
from the controls, monkeys 13 and 20, respectively. These values
were well within the range usually seen for SIVmac239 infection
(13). In contrast, no virus could be isolated even from
2,000,000 PBMCs from either of the 5G-infected monkeys 22 and 23, suggesting virtually complete virus clearance at this stage (8 weeks
p.i.). However, we did recover the viruses at 16 weeks from 2,000,000 (monkey 22) and 1,000,000 (monkey 23) PBMCs. Sequencing of the
env genes from the recovered virus, encoding the V1 to V2
region, unequivocally demonstrated that the recovered viruses were
5G and not revertants. Thus, we concluded that 5G persisted,
albeit greatly limited in replication, without being cleared from the bodies.
Taken together, these results demonstrate that one or more of the five
N-glycans play a luxury function required to establish and maintain a
full chronic infection but are totally dispensable for primary acute infection.
Challenge infection of 5G-infected monkeys with wild-type
SIVmac239.
As shown in Fig. 2,
another set of thee monkeys (7, 12, and 26), which
received 100 TCID50 of 5G, displayed essentially the
same replication pattern as seen for the two 5G-infected animals
shown in Fig. 1. Probably because of the outbred nature of the monkey
populations used, we sometimes observed a 5G-type replication
pattern in about 30% of the monkeys even following infection with
SIVmac239 (1). However, it has to be emphasized that with
the wild type we have never experienced such low levels of viremia in
all five arbitrarily chosen monkeys as seen for the 5G mutant. The
above three 5G-infected animals (7, 12, and 26) were
challenged with 500 TCID50 of SIVmac239 at 48 weeks after
the initial 5G infection and then assayed for plasma viral loads.
Remarkably, no viremia was detected during the observation period
starting at 2 weeks following challenge for two (7 and 26)
of the monkeys, and only transient viremia of a marginal level for the
remaining one was found at 2 weeks (Fig. 2). Serving as controls in
this challenge experiment were the two naive monkeys (13 and
20), which were described above and are shown in Fig. 1. A
comparison with these naive cases (Fig. 1) indicated that the immunity
induced by 5G was potent enough to contain the viral load in the
acute phase from as much as about 10,000,000 to 1,000 copies/ml or
less.
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FIG. 2.
Challenge infection of 5G-infected monkeys with
wild-type SIVmac239. Three animals (7, 12, and 26) were
initially infected with 100 TCID50 of 5G. After 48 weeks, they were challenged with SIVmac239 at an input dose of 500 TCID50. Plasma viral loads were determined throughout the
period of the initial infection and the challenge infection. The time
of challenge infection is indicated by an arrow.
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PBMCs were obtained from the monkeys 2 weeks before and at various
times after the challenge. They were dispensed into 10 tubes so that
each tube contained 50,000 cells, and the viral DNA encoding the V1 to
V2 region was amplified. When successfully amplified, the DNAs were
sequenced to determine whether they were of 5G or wild-type origin.
As shown in Table 1, proviral sequences could be amplified in 3, 5, and 2 of the 10 tubes for monkeys 7, 12, and 26, respectively, which altogether contained 500,000 PBMCs in
total, 2 weeks before challenge. Without exception, the mutations
introduced to remove the glycans were retained, and no additional
mutations were found in any of the amplified sequences. These data
indicated that 5G virus persisted over 46 weeks in all three monkeys
after the initial infection, though at a very limited level. After the
challenge, proviral sequences were almost consistently detected at
frequencies similar to those before challenge in monkeys 7 and 26. Their sequences were again of 5G origin. Thus, protection against
the wild type was apparently nearly complete in these two monkeys.
However, the results did not rule out the possibility that a transient
superinfection had taken place during the period from 0 to 2 weeks
after challenge, when no attempt at virus isolation was made.
Superinfection was not ruled out, either, at a level lower than was
detectable by the methods employed (use of 500,000 PBMCs and detection
level above 1,000 copies/ml). Indeed, superinfection was clearly
present in monkey 12, as 3 of the 7 sequences at week 2, all 10 at week
4, and 1 of the 5 at week 6 were of wild-type origin (Table 1). The
superinfection appeared to be transient and controllable, as no
wild-type clone became detectable by week 16. Taken together, these
results suggested that the 5G mutant could confer on rhesus macaques
not complete but remarkable protective immunity against SIVmac239.
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TABLE 1.
Detection frequency of 5G and SIVmac239 in PBMCs
from monkeys 7, 12, and 26 after challenge infection with SIVmac239
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Host responses following 5G infection.
Humoral responses
were compared between the 5G-infected (22 and 23) and
wild-type-infected (13 and 20) monkeys by assaying binding
ELISA antibody titers to wild-type SIVmac239 virion proteins and
neutralizing antibody titers against both the 5G and wild-type
viruses. As shown in Fig. 3A and B, no
appreciable difference was seen between the two monkey groups in either
the developmental sequence or the maximum titer of binding antibody.
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FIG. 3.
Anti-SIV ELISA. The OD492 was used as a
relative measure of antibody titer. (A) 5G infection. (B) SIVmac239
infection. (C) Before and various times after challenge infection of
5G-infected animals with SIVmac239 (arrow).
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We used essentially the same protocol for the neutralization assay as
used in previous macaque infection studies with deglycosylated SIVs
(19, 26), which involved infection of CEMx174 cells
containing a SEAP gene under the control of a Tat-responsive LTR. As
shown in Table 2, both of the
5G-infected monkeys displayed considerably high neutralization
titers (100 to 400) to the homologous virus at 20 weeks. Neutralizing
activity became detectable at 4 or 8 weeks p.i. in these animals (Table
2), before the time that set points were reached (Fig. 1). Sera from
the same animals possessed no appreciable neutralization titer against
the counterpart SIVmac239. Both of the SIVamc239-infected monkeys
displayed low levels of neutralization against the homologous and
heterologous strains for up to 20 weeks. These results may suggest that
greater containment of 5G was due in part to better humoral response
to the homologous virus.
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TABLE 2.
Neutralizing titers in rhesus macaques infected with
SIVmac239 (13 and 20) or 5G (22 and 23) against homologous and
heterologous viruses
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We then compared CTL responses against Gag-Pol, Nef, and Env from both
5G and wild-type SIVmac239 between the monkeys infected with 5G
(22 and 23) and those infected with the wild type (13 and 20). We found no temporal or quantitative
difference in the CTL response patterns between the two monkey groups.
However, slight increases (5 to 9%) in lysis were seen against 5G
Env compared to wild-type Env when effector cells from both
5G-infected and wild-type-infected monkeys were used (data not shown).
Host responses following challenge infection.
To find the
immunological correlates of protection induced by 5G, ELISA antibody
titers, neutralization antibody titers, and CTL responses were measured
before and after challenge infection of 5G-infected monkeys 7, 12, and 26 with wild-type SIVmac239. As shown in Fig. 3C, upon
challenge, ELISA titers immediately doubled in monkey 12, while no such
vigorous secondary response was seen for either 7 or 26. These results
were in agreement with the fact that transient superinfection with the
wild type was clearly detectable in monkey 12 but not in the other two
(Table 1). As shown in Table 3,
neutralization antibodies against the homologous strain were present in
two of the three 5G-infected monkeys at a relatively high (200, monkey 7) or marginal (25, monkey 12) level and undetectable in monkey
26 2 weeks before challenge infection. The levels of neutralization
were not much altered after SIVmac239 challenge in any monkey. On the
other hand, neutralization activities against the challenge virus
SIVmac239 were undetectable not only before but also after challenge
for up to 6 weeks (Table 3). These results strongly suggested that the
striking containment of the challenge virus was not due to neutralization antibodies.
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TABLE 3.
Neutralizing titers against SIVmac239 and 5G in rhesus
macaques 7, 12, and 26 infected with 5G and challenged with
SIVmac239
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Figure 4 compares the CTL responses
against multiple antigens following challenge with SIVmac239 between
naive and 5G-infected monkeys. In naive control animals 13 and 20, CTL against all these targets became detectable 4 weeks after challenge
and persisted for 20 weeks (Fig. 4A). CTL activities were undetectable
immediately before challenge for all three 5G-infected monkeys 7, 12, and 26 (Fig. 4B). However, it was remarkable that CTL became
clearly detectable by 2 weeks after challenge in all of them, 2 weeks earlier than in control monkeys (Fig. 4B). Moreover, the titers were
obviously higher in the former than in the latter, and remained so up
to 4 weeks. These results strongly suggested that 5G had mounted
sufficient memory to recruit vigorous secondary CTL responses against
multiple antigens quickly upon challenge infection.
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FIG. 4.
CTL responses at various times after infection of naive
monkeys with SIVmac239 (A) and at 2 weeks before and various times
after challenge (pc) of 5G-infected monkeys with SIVmac. Wild-type
Env was used.
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Superinfection is a prerequisite of immunological secondary responses.
Thus, the strong CTL responses observed here for all three
5G-infected monkeys support the above-described notion that we have
failed to detect actual superinfection for two of the monkeys (7
and 26). The CTL responses tended to decline rapidly in all
three immune monkeys by 20 weeks (Fig. 4B), suggesting efficient
control of the superinfecting viruses before 20 weeks.
| |
DISCUSSION |
Our deglycosylated mutant-5G retained full replication ability
in rhesus macaques in the primary acute phase, but its set point and
subsequent replication in the chronic period were greatly, though not
completely, contained (Fig. 1 and 2). Kimata et al. compared the
phenotypes in macaques and the Env sequences of a pair of strains,
SIVMneCL8 and its derivative, SIVMne35wkSU (4, 14, 27).
The former was found to be strongly contained, just like our 5G, in
the chronic phase, whereas the latter was as active as SIVmac239. The
former raised appreciable neutralization titers against the homologous
strain, whereas the latter failed to do so. These phenotypic
differences were closely related to the absence (former) or presence
(latter) of glycans in the V1 region. Thus, this pair of strains are
closely similar to our pair, 5G and SIVmac239. Reitter et al.
created a series of mutants with two glycans deleted in the V1 region
and between the V1 and V2 regions (25, 26). These mutants
were also attenuated in the chronic phase and induced extremely high
titers of neutralization antibodies. Probably because of such high
titers, the antibodies were able to neutralize not only the homologous
deglycosylated mutants but also the fully glycosylated wild type.
Moreover, when they reverted to greater virulence during the infection,
they always mutated to restore the glycans (26). Selection
of neutralization resistance of a simian-human chimeric virus in vivo
was found to occur by acquisition of N glycans in V1 and V3
(5). Thus, it now appears to be firmly established that
SIV and HIV strains with fewer glycans are better controlled and elicit
better humoral response.
As noted above, acquisition of N-glycans and reversion to a more
pathogenic phenotype were frequently seen for the mutants with one or
two glycans deleted in the region encompassing the V1 to V2 region
(26). This was not seen for 5G during the entire observation period. This stability was attributable to mutation at as
many as five sites. Reitter et al. also generated a mutant with five
potential N-glycosylation sites silenced (25, 26). According to our criteria, four of the sites are neutral, little affecting replication ability, if silenced individually, while the
remaining one did not appear to be actually glycosylated
(22). Significant attenuation of this mutant at acute
phase (a reduction in the peak titer of 10- to 100-fold)
(26) suggests that simultaneous deletion of four neutral
sites somehow impairs replication in vivo. Robust replication in acute
phase of 5G may be related to the fact that two (460 and 479) of the
sites deleted are downmodulating, in that removal of either greatly
enhances infectivity (22).
Strikingly low set points and strong containment in the chronic phase
were the characteristics shared by these multiple deglycosylation mutants. The better humoral responses to deglycosylated mutants may
be responsible. This view may be supported by detection of neutralizing
antibodies to 5G before set points were reached. In another case,
however, the set point appeared to be reached well before
neutralization antibodies became detectable (26), suggesting other responsible factors. Specific CTLs and helper T cells
could be significantly involved, as was seen in controlling wild-type
virus replication (2, 3, 21). However, as noted above, we
observed little difference in CTL response patterns between 5G- and
wild-type-infected monkeys. Alternatively, the apparently better
containment of deglycosylated mutants might have little to do with host
responses but could be caused by some impairment of functional and
structural integrity of Env due to removal of multiple glycans.
The 5G mutant was regarded as a novel class of live attenuated
vaccine because it was able to elicit not complete but remarkably potent protective immunity in rhesus macaques against wild-type SIVmac239. The protection appeared to correlate with strong secondary CTL responses in the absence of neutralizing antibodies, joining a list
of accumulating data that indicate a major role for CTL as an
immunological correlate of protection by live SIV and HIV vaccines. In
view of these previous data (8, 20, 28; for a review, see
reference 12), CTL responses not to Env but to multiple
other antigens, including Gag-Pol and Nef, could be important for the
good protective immunity elicited by 5G.
Without doubt, T-cell precursor frequencies depend on the initial viral
load or burst size. They may be maintained at fairly stable levels in
an antigen-independent manner (10, 15). However, more
recent reports provided evidence suggesting that a low level of viral
persistence is critical for maintaining the functional status of immune
memory cells even in mice acutely infected with and then rendered
immune to certain viruses, such as lymphocytic choriomeningitis virus
(6 and references therein). Thus, infection of monkeys
with 5G is closely similar to lymphocytic choriomeningitis virus
persistence in immune mice, suggesting that the robust primary replication followed by a marginal level of persistent infection in the
chronic phase underlie the strong secondary CTL responses against
multiple SIV antigens.
We thank R. Desrosiers and R. Means for providing CEMx174 SEAP
cells and for technical advice on the neutralization assay and D. L. Panicali (Therion Biologics) for providing the recombinant VV
expressing SIVmac239 Env, SIVmac251 Gag-Pol, and SIVmac239 Nef and the
parental virus (NYBCH strain).
This work was supported by AIDS research grants from the Ministry of
Health, Labour and Welfare, the Health Sciences Foundation, the
Organization for Pharmaceutical Safety and Research, and the Ministry
of Education, Science, Sports and Culture in Japan.
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Abstract
Introduction
