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Journal of Virology, February 2001, p. 1507-1515, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1507-1515.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Persistence of Pathogenic Challenge Virus in
Macaques Protected by Simian Immunodeficiency Virus
SIVmac
nef
Emmanuel
Khatissian,1,*
Valérie
Monceaux,1
Marie-Christine
Cumont,1
Marie-Paule
Kieny,2
Anne-Marie
Aubertin,2 and
Bruno
Hurtrel1
Unité d'Oncologie Virale, Institut
Pasteur, 75015 Paris,1 and INSERM U74,
Université Louis Pasteur, 67000 Strasbourg,2 France
Received 17 April 2000/Accepted 25 October 2000
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ABSTRACT |
Live attenuated simian immunodeficiency virus (SIV) is the most
efficient vaccine yet developed in monkey models of human immunodeficiency virus infection. In all successful vaccine trials, attenuation was achieved by inactivating at least the nef
gene. We investigated some virological and immunological
characteristics of five rhesus macaques immunized with a
nef-inactivated SIVmac251 molecular clone
(SIVmac251
nef) and challenged 15 months later with the
pathogenic SIVmac251 isolate. Three animals were killed 2 weeks
postchallenge (p.c.) to search for the challenge virus and to assess
immunological changes in various organs. The other two animals have
been monitored up for 7 years p.c., with clinical and nef
gene changes being noted. The animals killed showed no increase in
viral load and no sign of a secondary immune response, although the
challenged virus was occasionally detected by PCR. In one of the
monkeys being monitored, the vaccine virus persisted and an additional
deletion occured in nef. In the other monkey that was
monitored, the challenge and the vaccine (nef) viruses were both detected by PCR until a virus with a hybrid nef
allele was isolated 48 months p.c. This nef hybrid encodes
a 245-amino-acid protein. Thus, our results show (i) that monkeys were
not totally protected against homologous virus challenge but controlled
the challenge very efficiently in the absence of a secondary immune response, and (ii) that the challenge and vaccine viruses may persist
in a replication-competent form for long periods after the challenge,
possibly resulting in recombination between the two viruses.
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INTRODUCTION |
Experimental infection of macaque
with the simian immunodeficiency virus (SIV) is considered to be the
best model of human immunodeficiency virus (HIV) infection in humans.
SIV induces an AIDS-like disease in macaques and is therefore a useful
tool for the development of AIDS vaccines. Numerous approaches have been tried, including the use of whole inactivated viruses, recombinant proteins, nude DNA, and viral vectors alone or in combination, but
vaccines have to date been largely unsuccessful at inducing long-term
protection against heterologous challenge viruses in macaques (for a
review, see reference 28). Currently, the most successful
vaccines tested in the macaque model use a live attenuated SIV. The
first demonstration of protection provided by an attenuated virus
against challenge with a pathogenic virus (15) has been confirmed and extended by many authors (3, 14, 47, 63, 67,
71). Protection is inversely correlated with the level of
attenuation of the vaccine virus and requires several months to
establish (39, 71). The attenuated virus induces
protection against challenge with virus-infected cells and cell-free
virus administered by the intravenous or mucosal route (3,
45). However, the mechanisms underlying this protection are
still unclear. All live attenuated viruses that have been shown to
induce protection following vaccination carry at least an altered
nef gene.
In vitro, the Nef proteins of both HIV and SIV have several different
functions. Nef has been reported to down-regulate the surface
expression of CD4 (1, 21, 56) and the cell surface expression of class I major histocompatibility complex, preventing the
recognition and lysis of infected cells by cytotoxic lymphocytes (11, 60). Nef may also interact with a variety of cell
proteins involved in the cellular transduction pathway, although the
effect of these interactions on the activation pathway is unclear
(5, 25, 57). Nef also increases virion infectivity by
acting at the stage of particule production to increase the efficiency
of reverse transcription (RT) that immediately follows viral entry (2, 10, 59).
Nef is not essential for in vitro replication. However,
nef-inactivated mutants of SIVmac replicate at a much lower
rate than the wild type and do not cause an AIDS-like disease in
juvenile or adult animals (9, 32), although infection may
lead to disease development in newborn macaques (4, 72).
In humans, defective nef alleles have been characterized in
some long-term nonprogressor subjects (17, 33, 41, 55).
However, viruses in most of these subjects carry nef alleles
encoding a funtional protein as determined by single-cell infection or
CD4 down-regulation assays (27, 44). Moreover, truncations
in nef have also been identified in individuals with
progressive HIV disease, showing that nef defects are not
necessary sufficient to prevent progression toward AIDS (22,
65). Although the use of a live attenuated virus in humans is
currently inconceivable, trials of vaccination with such viruses in
monkeys may increase our understanding of the mechanisms involved in
protection against surperinfection and disease development.
In this study, we assessed the degree of protection against homologous
challenge with the pathogenic SIVmac251 isolate in five rhesus monkeys
vaccinated 15 months earlier with an attenuated nef-deleted
SIVmac251 virus. Three monkeys were killed 2 weeks postchallenge
(p.c.). The viral load in various organs and some immune
characteristics were evaluated for these monkeys. Two other monkeys
were studied for 7 years: we assessed the persistence of vaccine and
challenge viruses, changes in their nef genes, and long-term
clinical progression. We found that four of the five monkeys were not
totally protected but efficiently controlled the SIVmac251 challenge
virus in the absence of any signs of a secondary immune response. One
of the two monkeys monitored in the long term was protected against
challenge, and a new deletion has appeared in the nef region
overlapping the U3, long terminal repeat (LTR). In the other animal
monitored in the long term, both challenge and vaccine viruses
persisted and eventually a new hybrid virus (with a hybrid
nef gene) emerged, probably due to a recombination between
the two viruses.
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MATERIALS AND METHODS |
Viruses and animals.
The attenuated virus,
SIVmac251nef, was provided by M. P. Kieny
(Transgene, Strasbourg, France). It was derived from the SIVmac251 BK28
clone (35) by three modifications: (i) the premature stop
codon at position 8785 in the env gene was mutated to
restore a complete env open reading frame (ORF), (ii) the
nef initiator codon ATG was mutated to ACG at position 9059, and (iii) nucleotides 9225 to 9401 in the nef region, which
do not overlap either the 3' end of env or the U3 part of
the LTR, were deleted. All viruses were propagated on macaque
peripheral blood mononuclear cells (PBMC). The pathogenic SIVmac251
isolate provided by R. Desrosiers (16) was subjected to
titer determination in Chineese rhesus macaques (Macaca
mulatta) by intravenous inoculation. In a first experiment,
10-fold serial dilutions of the stock (8,000 50% tissue culture
infectious doses) were inoculated into groups of three macaques (1 ml
of 102 to 106 dilutions). Macaques were
determined to be infected after seroconversion and/or virus isolation
from PBMC. Since none of the monkeys inoculated with the
105 and 106 dilutions were infected, in a
second step macaques were inoculated with 1 ml of 104 and
105 dilutions and two intermediate dilutions to confirm and
determine the virus titer more precisely. Again macaques inoculated
with the 105 dilution remained uninfected. A 1-ml volume of
stock virus contained 4 × 104 50% animal infectious
doses (AID50) as determined by the VACMAN program, kindly
provided by J. Spouge (62).
Rhesus macaques were maintained in accordance with European guidelines.
Before inoculation, they were demonstrated to be seronegative for
simian T-cell leukemia virus type 1, simian retrovirus type 1 (type D
retrovirus), herpes B virus, and SIVmac. All the animals were
inoculated with cell-free virus by the intravenous route. Eight monkeys
were included in this study. Five were immunized with the attenuated
SIVmac251nef molecular clone (corresponding to 100,000 cpm in a reverse transcriptase assay). Fifteen months later, the five
immunized monkeys and the three naive monkeys used as controls were
inoculated with 10 AID50 of the pathogenic SIVmac251 isolate.
Serologic assays.
Plasma p27 Gag antigen levels were
determined using a specific SIVmac antigen capture enzyme-linked
immunosorbent assay (ELISA) (Coulter). The antibody response to SIV was
monitored by an HIV-2 ELISA (Elavia-II; Sanofi-Pasteur), which is
cross-reactive for SIV antibody. Serum samples were tested at serial
10-fold dilutions from 1:10 to 1:100,000.
Flow cytometry.
EDTA-treated blood was incubated for 15 min
with antibodies against CD4 (OKT4 [Ortho Diagnostic]) and CD8 (Leu-2a
[Becton Dickinson]), added at a 1:20 dilution. Erythrocytes were
lysed with Lyse & Fix reagents (Immunotech). The samples were washed three times in phosphate-buffered saline and fixed in
phosphate-buffered saline containing 1% paraformaldehyde. They were
analyzed by flow cytometry using a FACScan cytometer (Becton Dickinson).
In situ hybridization.
Hybridization was performed as
previously described with a 35S-labeled RNA nef
probe derived from the nef SIVmac142 sequence (9).
nef amplification and sequencing.
RNA was
extracted from serum as follows. First-time-thawed serum was
centrifuged at 15,000 rpm (20,627 × g) for 2 h to
pellet the virions. The pellet was resuspended in 300 µl of lysis
buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA, 0.1% sodium
dodecyl sulfate, 1 mg of proteinase K per ml) and incubated at 56°C
for 30 min. The mixture was extracted twice with phenol and chloroform isoamyl alcohol (24:1), a glycogen carrier (40 µg/ml) (Roche), and a
synthetic 7.5-kb RNA (1 µg/ml) (Gibco-BRL) were added. RNA was
precipitated with ethanol and pelleted by centrifugation at 15,000 rpm
(20,627 × g) for 30 min. Total RNA from organs and total cellular DNA from PBMC were extracted using standard methods. For
quantitative RT-PCR, a series of 1:2 dilutions of RNA in
diethylpyrocarbonate-treated water was used for RT.
For reverse transcription, 0.25 µg of total RNA was reverse
transcribed with 50 U of reverse transcriptase (Moloney murine
leukemia
virus superscript, Gibco-BRL) in a mixture containing
1× PCR buffer II
(Perkin-Elmer), 5 mM MgCl
2, 1 mM each deoxynucleoside
triphosphate, 2.5 µM oligo-d(T)
16 and 20 U of RNase
inhibitor
(RNAguard; Pharmacia). The reaction was conducted at 42°C
for
30 min followed by heat inactivation at 95°C for 5
min.
For quantitative nested PCR, a known amount of DNA competitor was added
to the cDNA. The following
nef primers were used for
detection of the
nef sequence: Preco
(5'-CAGAGGCTCTCTGCGACCCTAC-3')
and K3
(5'-GACTGAATACAGAGCGAAATGC-3') in the first amplification,
and K1 (5'-TGGAAGATGGATCCTCGCAATCC-3') and A2
(5'-GGACTAATTTCCATAGCCAGCCA-3')
in the second amplification.
DNA was subjected to 35 cycles of
amplification in a volume of 100 µl
containing 1× PCR buffer II,
0.2 mM each deoxynucleoside triphosphate,
2 mM MgCl
2, 0.15 µM
each external primer, and 2.5 U of
AmpliTaq (Perkin-Elmer). For
the second PCR, 5 µl from the first
amplification was subjected
to 25 cycles of amplification in a total
volume of 100 µl of PCR
mixture containing the internal primers.
Under these conditions,
it was possible to detect 25
nef in
vitro transcripts. Amplification
products were analyzed by
electrophoresis in a 2.5% agarose gel
stained with ethidium bromide.
For quantitative PCR, the amplified
competitor was distinguished by its
specific size. The competitor-template
equivalent point was determined
by visual examination of the photograph
of the
gel.
Amplified fragments were inserted into the pGEM-T Easy vector (Promega)
for sequencing by Genome Express (Grenoble, France),
or used for
coupled transcription-translation with the TNT-coupled
reticulocyte
lysate system (Promega) as specified by the
manufacturer.
Virus recovery from PBMC.
PBMC from heparinized blood were
separated on a Ficoll density gradient. The cells were washed twice in
RPMI 1640, and CD8+ PBMC cells were removed by
immunomagnetic separation with anti-CD8 Dynabeads (Dynabeads M-450 CD8;
Dynal) as specified by the manufacturer. CD8+-depleted PBMC
were resuspended in RPMI complete medium (RPMI 1640 supplemented with
10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U of
penicillin per ml, and 100 µg of streptomycin per ml) and stimulated
by incubation with phytohemagglutinin (5 µg/ml) (Sigma) and
recombinant interleukin-2 (20 U/ml) (Roche) for 48 h. CEMx174
cells were then added to the culture. SIV-infected cultures were
diagnosed by PCR detection of provirus and assessment of syncytium formation.
| |
RESULTS |
Vaccination with SIVmac251nef and challenge with the
SIVmac251 isolate.
Five monkeys were vaccinated by intravenous
injection with a SIVmac251 molecular clone attenuated by inactivation
of the nef gene (SIVmac251nef). The primary
infection of three monkeys (52168, 90141 and 90154) has been previously
reported (9). Briefly, the major feature of infection with
this attenuated virus was a much lower viral load than obtained in
infection with virus containing a full-length nef. During
the 15-month vaccination phase, no clinical signs were displayed by the
monkeys. Around the time of challenge, anti-SIV immunoglobulin G (IgG)
persisted at the level reached 2 months postinfection, with a titer of
1,000 to 10,000, and the virus was sometimes detected in the blood by PCR. Thus, the vaccine virus persisted at a low level, together with a
vigorous specific immune response.
Vaccinated monkeys and three naive monkeys used as a control, were
challenged intravenously with 10 AID
50 of the pathogenic
SIVmac251 isolate. Table
1 summarizes the
vaccination status
and duration of follow-up of the monkeys used in
this study. Three
vaccinated monkeys (52168, 90141, and 90154) and one
naive monkey
(92409) were killed 2 weeks p.c. for analysis of the
distribution
of virus in various organs including the lymph nodes (LN),
spleen,
and brain. Two other immunized monkeys (49848 and 49851) and
two
naive challenged monkeys (92418 and 92428) were observed over
time,
with viral changes and clinical progression being monitored.
Viral load and immune response analyses for animals killed 2 weeks
p.c.
Viral load in the serum was evaluated after challenge by
determining the levels of Gag p27 antigen and virion-associated viral RNA. In all vaccinated monkeys tested, p27 antigen levels, determined by ELISA, were similar to background levels. In contrast, p27 antigen
was detectable in the serum of the infected naive monkey (92409) from
day 7 onward. Viral RNA was detected on one occasion in the sera of the
three vaccinated monkeys killed 2 weeks p.c. (Table
2). Only the deleted form of
nef, corresponding to the vaccine virus, was detected and
was present at less than 300 copy equivalents per ml. In contrast, the
wild-type nef sequence was detected from day 7 onward and
reached 2.5 × 105 copy equivalents in the serum of
the infected naive monkey at the time when it was killed. Given the
sensitivity of this method, these data indicate that particles of the
challenge virus did not circulate in the blood of vaccinated monkeys
within the 2 weeks p.c. They also show that challenging the vaccinated
monkeys with a pathogenic isolate did not stimulate replication of the vaccine virus resident in the blood.
Viral replication was also sequentially investigated in LN from the
three vaccinated animals (52168, 90141, and 90154) and
the one naive
animal (92409) killed 2 weeks p.c. The spleen and
brain were also
collected at the time of death. We carried out
in situ hybridization
with a
nef probe that detects the transcripts
of both the
vaccine and challenge viruses. Positive signals were
very rarely
detected in the LN of vaccinated challenged monkeys
collected on days
0, 7, and 14 or in the spleen and brain (Fig.
1). In contrast, in the naive monkey
(92409), productively infected
cells were detected in the LN from day 7 onward and in the spleen
at the time of death, albeit at a lower
frequency than in the
LN. To increase sensitivity and to discriminate
between challenge
and vaccine virus mRNA, the viral load in organs was
also investigated
by RT-PCR, amplifying the
nef region
(Table
3). As with the in
situ approach,
no virus was detected in the brain. In the LN and
spleen, transcripts
of vaccine virus were detected in the three
vaccinated monkeys within 2 weeks of challenge. Competitive RT-PCR,
performed with 0.25 µg of RNA
from these samples, showed that
positive signals corresponded to a
maximum value of 200 copy equivalents
of viral mRNA. In contrast, in
the naive monkey (92409), 10,000
copy equivalents were detected 2 weeks
p.c. in LN whereas only
700 copy equivalents were detected in the
spleen. This is consistent
with the lower viral load detected in the
spleen by in situ hybridization.
Challenge virus transcripts were also
detected 2 weeks p.c. in
the LN and spleens of two (90141 and 90154) of
the three vaccinated
monkeys.
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FIG. 1.
Comparison between LN collected at the time of challenge
and 2 weeks after challenge. LN were obtained at the time of SIVmac251
challenge and 2 weeks p.c. from the three vaccinated monkeys (52168, 90141, and 90154) and the naive monkey (92409) killed for organ
analysis. Vaccine and challenge viruses were detected by in situ
hybridization with a 35S-labeled nef riboprobe.
Sections were stained with hematoxylin and eosin. Magnification,
×40.
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We also tested for the presence of viral sequences in PBMC by PCR
amplification of the
nef region. At one time point after
challenge, at least, the proviral sequence of the
nef
vaccine
virus was detected in two (52168 and 90154) of the three
vaccinated
monkeys (Table
3). In particular, the SIVmac251 challenge
virus
was detected in monkey 52168 1 week p.c. At this time point,
proviral
sequences became detectable in the PBMC of the naive monkey
(92409).
The detection of SIVmac251 viral transcripts in the LN and spleen of
two vaccinated monkeys (90141 and 90154) and of proviral
sequence in
the PBMC of the third (52168) indicates that these
monkeys were not
fully protected. However, viral replication did
remain very efficiently
controlled. Our data also show that challenge
of the vaccinated monkeys
was not followed by an increase in
nef viral
load.
We investigated whether the very efficient control of challenge virus
involved the development of a secondary immune response
by
studying some immunological characteristics for 2 weeks p.c.
In
contrast to what was observed in naive monkeys, the challenge
of
vaccinated monkeys with the pathogenic SIVmac251 virus did
not
induce any sign of immune reactivation in the LN according
to the
following criteria: (i) no extension or decrease of the
preexisting
germinal centers already well developed before challenge
(Fig.
1), (ii)
no change in mRNA level for the cytokines or chemokines
tested
(interleukin-2 [IL-2], IL-10, gamma interferon, RANTES,
macrophage
inflammatory protein 1
[MIP-1
], or MIP-1
) by RT-PCR
(data
not shown) and (iii) no secondary IgG response to whole
viral antigens
during the 2 weeks following challenge (Fig.
2).
Therefore, challenging vaccinated
monkeys with the pathogenic
virus did not seem to induce a secondary
immune response and the
preexisting immunity was sufficient to control
any replication
of the pathogenic challenge virus.
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FIG. 2.
Antibody response after challenge. Anti-SIV IgG antibody
titers were determinated by ELISA in sera of the group I monkeys (see
Table 1) challenged with the pathogenic SIVmac251 isolate and killed 2 weeks later. The titer is expressed as the optical density (492 nm)
multiplied by the final dilution before extinction of the specific
signal.
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Long-term persistence of challenge and vaccine viruses in
vaccinated monkeys.
We assessed the persistence of vaccine and
challenge viruses in the two vaccinated monkeys (49848 and 49851)
monitored up for 7 years after being challenged with the pathogenic
SIVmac251 isolate. During this period, these monkeys showed no clinical signs or decrease in CD4+ cell counts. As controls, two
naive animals were also challenged (92418 and 92428). As expected, the
unvaccinated monkeys tested positive for the virus by PCR at all time
points after 1 week p.c. Both these monkeys also showed a severe
decline in CD4+ cell counts, and they died at 21 and 31 months p.c.
PBMC were regularly collected from vaccinated and challenged monkeys.
DNA was isolated from these cells and used for the nested
PCR
amplification of
nef sequences. Proviral sequences were
frequently
detected during the 62 months of PCR follow-up (Table
4). The
only form detected for the PBMC
of monkey 49848 was the same size
as that of the
nef
amplicon control, suggesting that the vaccine
virus had persisted
without major modifications in the
nef region.
In contrast,
for monkey 49851, two forms were initially detected,
of the same sizes
as the vaccinal and challenge
nef amplicons,
and then at 56 months p.c. an amplicon of intermediate size was
detected.
We investigated whether the proviral sequence amplified from monkeys
monitored in the long term corresponded to an efficient
replicating
virus. We tried to isolate viruses by coculture of
activated PBMC with
CEMx174 cells. Cocultures starting with PBMC
from which
CD8
+ cells were or were not depleted were established 48, 56, 62,
and 76 months p.c. Isolation was successful only from
CD8
+-depleted PBMC, illustrating the well-known inhibitory
role of
CD8
+ cells in viral replication. For monkey 49851, virus was isolated
from cocultures established 48 and 76 months p.c.
whereas isolation
was successful only 76 months p.c. for monkey 49848. The time
from the beginning of coculture to the appearance of the
syncytia
in CEMx174 culture was 3 weeks for monkey 49848 for coculture
76 months p.c. and 5 and 3 weeks for monkey 49851 for coculture
48 and
76 months p.c., respectively. When the same experiment
was conducted
with CD8
+-depleted PBMC from the two control monkeys (92418 and 92428)
in the asymptomatic phase, syncytia were visible within 1 to
3
weeks.
We characterized the
nef allele of the viruses isolated from
vaccinated and challenged monkeys by PCR amplification of the
complete
nef gene plus the 3' end of the LTR U3 region from DNA
extracted from the culture (see Fig.
3 for the locations of the
primers
and the genetic organization of this region). These PCR
products, named
nef-U3 LTR, were cloned and
sequenced.
Changes in nef sequences in the vaccinated and
challenged monkeys.
Figure 3 shows
the nef-U3 LTR region of the virus isolated from the PBMC of
monkey 49848, 76 months p.c. The original 175-bp deletion was
unaffected, but two important modifications were observed. The first is
an additional 291-bp deletion in U3 beginning 43 bp downstream from the
polypurine tract and ending 78 bp upstream from the NF-
B site. The
second is the presence of a nef initiator codon, inactivated
in the original vaccine virus, suggesting a potential ORF encoding the
first 56 amino acids of Nef.
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FIG. 3.
Schematic representation of changes in nef
and U3 after challenge with the SIVmac251 isolate in macaques
vaccinated with the live attenuated virus SIVmac251 nef.
Nucleotides are numbered according to the SIVmac251 BK28 nef
gene from which the vaccine virus was derived (35). Since
the challenge virus is an uncloned virus, we have represented the
SIVmac239 molecular clone. Arrows at the top indicate the binding sites
of the primers used for PCR amplification.
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The
nef-U3 LTR PCR product obtained from the coculture with
the PBMC of monkey 49851 collected 48 months p.c. was reproducibly
found to have a size intermediate between those of the forms of
nef of the vaccine and challenge viruses. Sequence analysis
suggested
that this
nef gene is a hybrid between the vaccine
and challenge
virus forms of
nef. Figure
3 presents a
comparison of the
nef-U3
LTR region of the hybrid with that
of the vaccine virus. This
hybrid
nef gene has an initiator
codon, and the original deletion
has been reduced from 175 to 48 bp by
insertion of the wild-type
sequence and an additional 6-bp deletion in
the U3 region. In
addition, the insertion of a thymine at position 699 (relative
to the
nef of SIVmac251 BK28) results in a
frameshift, changing
the C-terminal 15 amino acids of Nef into a
31-amino-acid extremity
identical to that encoded by SIVmac239. Thus,
the hybrid ORF potentially
encodes a 245-amino-acid Nef protein.
Finally, a third Sp-1 site
absent from the vaccine virus is also
present, as in
SIVmac239.
We investigated whether this
nef ORF actually encodes a
protein by performing a coupled transcription-translation assay using
the cloned
nef-U3 LTR PCR product. Cloned
nef-U3
LTR PCR products
from SIVmac251 BK28 and vaccinal
SIVmac251
nef clones were used
as controls. The
transcription-translation assay resulted in the
synthesis of a protein
with an apparent molecular mass of 28 kDa,
close to that of the
SIVmac251 BK28 Nef protein (Fig.
4). This
result is consistent with the removal of 16 amino acids created
by the
48-bp deletion, compensated by the restoration of a full
coding
C-terminal extremity generating 16 additional amino acids.
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FIG. 4.
Coupled transcription-translation assay of
nef alleles. Methionine-labeled Nef protein was analyzed by
electrophoresis in a 12% acrylamide denaturing gel. Lanes (from left
to right): SIVmac251 nef, SIVmac251 BK28 nef,
SIVmac nef isolated from monkey 49851, molecular mass
markers (M.W.), negative control with T3 and T7 RNA polymerases and no
DNA, positive controls from the kit, under the control of the T3 and T7
promoters, respectively.
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A
nef-U3 LTR PCR product of intermediate size was also
detected in PBMC collected 56 and 62 months p.c. (Table
4). The same
48-bp deletion was observed in the PCR product obtained 62 months
p.c.
and in the virus isolated 76 months p.c. These results suggest
that the
hybrid virus emerged between month 32 p.c. (i.e., the
last point
at which vaccine virus was detected in PBMC) and month
48 p.c.
(the time point at which the
nef hybrid virus was first
isolated). After its initial detection, this hybrid form was the
only
form of
nef detected in the PBMC of monkey
49851.
| |
DISCUSSION |
This study was designed to assess the degree of protection
provided by vaccination with a live attenuated
SIVmac251nef virus. Five rhesus monkeys vaccinated 15 months previously were challenged with a low dose (10 AID50) of the pathogenic SIVmac251 isolate. Our PCR results
indicate that at least four of these monkeys were not totally protected
against the challenge virus. In this kind of vaccination approach,
protection is greater the more similar the vaccine and challenge
viruses, requires the vaccine virus to persist, and takes several
months to become fully established (12, 71). Taking into
account the duration of vaccination, the persistence of the vaccine
virus, and its similarity to the challenge virus, our data suggest that
even optimal vaccination conditions do not systematically induce total
protection against a homologous challenge virus. Several recent studies
have reported that vaccination with a nef SIVmac virus
may not provide protection against challenge with a heterologous
pathogenic virus or against a homologous virus if the duration of
vaccination is too short (12, 38, 70). Moreover,
superinfection with the pathogenic challenge virus may lead to
CD4+ cell depletion. In this study, in contrast, the
pathogenic challenge virus was very efficiently controlled, since no
increase in the load of the resident vaccine virus and no decrease in
the CD4+ cell count were observed in the monkeys observed
for 7 years. These data suggest that in our system, vaccination does
not protect against superinfection but does protect against disease development.
We observed no signs of a secondary immune response following the
pathogenic challenge in the monkeys killed 2 weeks p.c. We therefore
conclude that the preexisting immunity was sufficient to control the
challenge virus and thus prevented the development of a secondary
immune response. Generally, inefficient control of challenge virus is
responsible for the development an anaemestic response
(47) that may also permit reactivation of the resident vaccine virus (49).
The mechanisms underlying this control are unknown. A nonimmune
mechanism such as receptor interference is unlikely, because vaccinated
monkeys are also protected against challenge with a SIV pseudotype
carrying the amphotropic envelope of the murine leukemia virus
(24). However, as far as immune mechanisms are considered,
protection against a SIV carrying an amphotropic or HIV-1 envelope
excludes a major role for neutralizing antibodies (7, 19,
61). Several studies have implicated the CTL response in the
control of SIV infection (31, 42, 51, 58). However, in
monkeys vaccinated with the attenuated SIVmac251 32H(C8) virus, the
partial depletion of CD8+ cells before challenge did not
abolish protection against SIVmac251 32H, the pathogenic isolate
corresponding to the vaccine virus (64).
Few studies have focused on the long-term effects of the persitence of
vaccine and/or challenge viruses. We therefore monitored two vaccinated
monkeys for 6 and 7 years, studying changes in the viruses they carried
and their clinical progression. Only vaccine sequences were detected in
the PBMC of monkey 49848 throughout the 62 months of follow-up p.c.
However, it could not be strictly concluded that this monkey has been
totally protected against challenge, because the presence of SIV DNA
was not investigated in LN and in two (90141 and 90154) of the three
monkeys killed at 2 weeks p.c. the challenge virus was detected in LN
but not in PBMC. Moreover, McChesney et al. have recently shown that, in occult infection following inoculation with a low dose of SIVmac251 by the intravaginal route, SIV DNA was detected more frequently in LN
than in PBMC years after inoculation (43). Virus isolated 76 months p.c. showed, in addition to the original deletion, a 290-bp
deletion in the overlaping nef-U3 region. This deletion did
not affect the sequence elements important for viral replication and
transcription (i.e., the polypurine tract and its immediate downstream
sequences, the enhancer region just upstream from the NF-B, the
NF-B and Sp-1 binding sites, and the TATAA box). We observed the
same features in unchallenged macaques infected with SIVmac251nef (data not shown). These data are consistent
with previous in vitro and in vivo observations for
SIVmac239nef-infected monkeys (12, 29, 34,
50). Thus, in the absence of a functional nef,
certain regions overlapping nef and U3 are lost, suggesting that these regions are dedicated solely to the encoding of Nef. Since
one inner primer bound to the sequence located in the additional deletion, PCR analysis could not be used to detect the virus in PBMC
before its isolation.
In the second challenged and monitored monkey (49851), both challenge
and vaccine virus sequences were frequently detected. The virus
isolated 48 months p.c. carried a hybrid sequence, intermediate between
those of the vaccine and challenge viruses in the nef-U3 LTR
region, encoding a 245-amino-acid protein. This nef hybrid gene did not result from an in vitro artifact such a recombination during PCR or growth on CEMx174, since it was again directly amplified by PCR from PBMC 62 and 76 months p.c. and reisolated at month 76 p.c. Thus, the nef hybrid virus isolated 48 months p.c. was actually the major form in vivo. The emergence of a SIV strain with a
hybrid nef gene raises questions about its origin and the reasons for its positive selection.
As regards its origin, there are two possible hypotheses: a spontaneous
48-bp deletion in the nef gene of the challenge virus or a
recombination event between the vaccine and challenge viruses. Many
studies have shown that there is strong pressure favoring the
maintenance of an intact nef gene in vivo. When first
isolated after in vitro amplification, both the SIVmac239 and SIVmac251 BK28 molecular clones showed defects in nef generated by
single point mutations (35, 52). However, following the
inoculation of monkeys with these viruses, the premature stop codon of
SIVmac239 reverted very rapidly and the frameshift in the SIVmac251
BK28 nef tended to disappear by means of a thymine insertion
(9, 20, 32, 36). In both cases, the restoration of a
full-length nef gene was associated with an increase in
virulence. The repair of a 12-bp deletion in the SIVmac251 32H(C8)
nef gene was also found to be associated with a reversion to
virulence (18, 54, 68). These observations indicate that
there is strong selection pressure in vivo, favoring the restoration of
an intact nef gene, unless nef is totally
inactivated. To our knowledge, Nef alterations have never been
associated with positive selection of the virus in vivo. Thus, it is
unlikely that a positive advantage is conferred in vivo by the
16-amino-acid nef deletion. Although we cannot formally
exclude the possibility of a spontaneous 48-bp deletion in the
challenge virus, we believe that the nef hybrid virus
results from recombination between the vaccine and challenge viruses, especially since the challenge and vaccine virus forms were
simultaneously detected several times before isolation of the hybrid virus.
The high recombination potential of retroviruses has been extensively
shown in vitro (26, 30, 73). Until recently,
recombinations in vivo between HIV or SIV were rarely described,
because for recombination to occur, individuals must be coinfected with
divergent strains and because most naturally occurring recombination
events would generate nonfunctional viruses, or at least viruses with reduced fitness, which are harder to identify. However, an increasing number of reports based on phylogenetic analysis have strongly suggested that recombination occurs in HIV-infected individuals (6, 8, 13, 40, 46, 48, 53, 66). SIVmac recombination in
macaques has been directly demonstrated by Wooley et al., by the
simultaneous inoculation of a naive monkey with two SIVmac239 viruses,
attenuated by different deletions (69). As early as 2 weeks after infection, the sequence of the full-length pathogenic SIVmac239 was the sequence predominantly detected, demonstrating that
recombination is of biological relevance in vivo.
Whatever its origin, the nef hybrid virus became the sole
form detected after its first identification, suggesting that it has a
selective advantage. The most obvious explanation for this positive
selection is that it has a higher intrinsic replication rate.
Curiously, in a single-cycle infectivity assay performed on sMAGI
cells, the nef hybrid virus was less infectious than even
the vaccine SIVmac251nef strain (data not shown),
suggesting that viral determinants other than nef may be
responsible for this in vitro phenotype. In addition, the in vitro
assay takes into account only some of the functions mediated by
nef during the early stages of the virus cycle in vitro, and
the significance of these functions in vivo is unclear. Thus, the data
obtained in vitro do not necessarily imply that the nef
hybrid allele is not functional in vivo. Indeed, several lines of
evidence led us to conclude that this allele has some functions in
vivo: (i) the deletion is short, does not map to a region known to have a critical function, and is restricted to a region described as disorder in HIV Nef (23, 37), and (ii) a nef
ORF is maintained. However, the 16-amino-acid deletion is unlikely to
cause an increase in replication in vivo. An alternative explanation
for the predominance of the nef hybrid virus is that it may
be able to escape more efficiently from immune control, regardless of
replication capacity. It is therefore intriguing that the
nef vaccine virus, despite its lower replication capacity
and greatly impaired potential to maintain a high viral load in naive
monkeys, is still codetected with the challenge virus several months
p.c. Finally, taking into account the low dose of inoculum and the very
efficient immune response, it is possible that defective viruses, which
constitute the majority of retroviral particles, are selected after
immune clearance. According to this hypothesis, the challenge and
vaccine viruses may coexist until a recombination event causes an
increase in viral fitness. It would be of value to inoculate monkeys
with this nef hybrid virus and to monitor them over time.
In conclusion, our results show (i) that monkeys were not totally
protected against homologous virus challenge but controlled the
challenge virus very efficiently in the absence of a secondary immune
response and (ii) that the challenge and vaccine viruses may persist in
a replication-competent form for long periods after the challenge,
possibly resulting in recombination between the two viruses.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the Agence Nationale de
Recherche sur le SIDA. E.K. was awarded a doctoral fellowship from SIDACTION.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité
d'Oncologie Virale, Département Rétrovirus, Institut
Pasteur, 28 Rue du Dr Roux, 75015 Paris Cedex 15, France. Phone: 33 (1)
40 61 32 65. Fax: 33 (1) 40 61 34 50. E-mail:
ekhatiss{at}pasteur.fr.
| |
REFERENCES |
| 1.
|
Aiken, C.,
J. Konner,
N. R. Landau,
M. E. Lenburg, and D. Trono.
1994.
Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain.
Cell
76:853-864[CrossRef][Medline].
|
| 2.
|
Aiken, C., and D. Trono.
1995.
Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis.
J. Virol.
69:5048-5056[Abstract].
|
| 3.
|
Almond, N.,
K. Kent,
M. Cranage,
E. Rud,
B. Clarke, and E. J. Stott.
1995.
Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells.
Lancet
345:1342-1344[CrossRef][Medline].
|
| 4.
|
Baba, T. W.,
Y. S. Jeong,
D. Pennick,
R. Bronson,
M. F. Greene, and R. M. Ruprecht.
1995.
Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.
Science
267:1820-1825[Abstract/Free Full Text].
|
| 5.
|
Bell, I.,
C. Ashman,
J. Maughan,
E. Hooker,
F. Cook, and T. A. Reinhart.
1998.
Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation.
J. Gen. Virol.
79:2717-2727[Abstract].
|
| 6.
|
Blackard, J. T.,
B. R. Renjifo,
D. Mwakagile,
M. A. Montano,
W. W. Fawzi, and M. Essex.
1999.
Transmission of human immunodeficiency type 1 viruses with intersubtype recombinant long terminal repeat sequences.
Virology
254:220-225[CrossRef][Medline].
|
| 7.
|
Bogers, W. M.,
H. Niphuis,
P. ten Haaft,
J. D. Laman,
W. Koornstra, and J. L. Heeney.
1995.
Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: consequences of challenge.
AIDS
9:F13-F18[Medline].
|
| 8.
|
Burke, D. S.
1997.
Recombination in HIV: an important viral evolutionary strategy.
Emerg. Infect. Dis.
3:253-259[Medline].
|
| 9.
|
Chakrabarti, L.,
V. Baptiste,
E. Khatissian,
M. C. Cumont,
A. M. Aubertin,
L. Montagnier, and B. Hurtrel.
1995.
Limited viral spread and rapid immune response in lymph nodes of macaques inoculated with attenuated simian immunodeficiency virus.
Virology
213:535-548[CrossRef][Medline].
|
| 10.
|
Chowers, M. Y.,
C. A. Spina,
T. J. Kwoh,
N. J. Fitch,
D. D. Richman, and J. C. Guatelli.
1994.
Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.
J. Virol.
68:2906-2914[Abstract/Free Full Text].
|
| 11.
|
Collins, K. L.,
B. K. Chen,
S. A. Kalams,
B. D. Walker, and D. Baltimore.
1998.
Hiv-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes.
Nature
391:397-401[CrossRef][Medline].
|
| 12.
|
Connor, R. I.,
D. C. Montefiori,
J. M. Binley,
J. P. Moore,
S. Bonhoeffer,
A. Gettie,
E. A. Fenamore,
K. E. Sheridan,
D. D. Ho,
P. J. Dailey, and P. A. Marx.
1998.
Temporal analyses of virus replication, immune responses, and efficacy in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine.
J. Virol.
72:7501-7509[Abstract/Free Full Text].
|
| 13.
|
Cornelissen, M.,
G. Kampinga,
F. Zorgdrager, and J. Goudsmit.
1996.
Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization.
J. Virol.
70:8209-8212[Abstract].
|
| 14.
|
Cranage, M. P.,
A. M. Whatmore,
S. A. Sharpe,
N. Cook,
N. Polyanskaya,
S. Leech,
J. D. Smith,
E. W. Rud,
M. J. Dennis, and G. A. Hall.
1997.
Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa.
Virology
229:143-154[CrossRef][Medline].
|
| 15.
|
Daniel, M. D.,
F. Kirchhoff,
S. C. Czajak,
P. K. Sehgal, and R. C. Desrosiers.
1992.
Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene.
Science
258:1938-1941[Abstract/Free Full Text].
|
| 16.
|
Daniel, M. D.,
N. L. Letvin,
N. W. King,
M. Kannagi,
P. K. Sehgal,
R. D. Hunt,
P. J. Kanki,
M. Essex, and R. C. Desrosiers.
1985.
Isolation of T-cell tropic HTLV-III-like retrovirus from macaques.
Science.
228:1201-1204[Abstract/Free Full Text].
|
| 17.
|
Deacon, N. J.,
A. Tsykin,
A. Solomon,
K. Smith,
M. Ludford-Menting,
D. J. Hooker,
D. A. McPhee,
A. L. Greenway,
A. Ellett,
C. Chatfield, et al.
1995.
Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.
Science
270:988-991[Abstract/Free Full Text].
|
| 18.
|
Dittmer, U.,
T. Nisslein,
W. Bodemer,
H. Petry,
U. Sauermann,
C. Stahl-Hennig, and G. Hunsmann.
1995.
Cellular immune response of rhesus monkeys infected with a partially attenuated nef deletion mutant of the simian immunodeficiency virus.
Virology
212:392-397[CrossRef][Medline].
|
| 19.
|
Dunn, C. S.,
B. Hurtrel,
C. Beyer,
L. Gloeckler,
T. N. Ledger,
C. Moog,
M. P. Kieny,
M. Mehtali,
D. Schmitt,
J. P. Gut,
A. Kirn, and A. M. Aubertin.
1997.
Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1.
AIDS Res. Hum. Retroviruses
13:913-922[Medline].
|
| 20.
|
Edmonson, P.,
M. Murphey-Corb,
L. N. Martin,
C. Delahunty,
J. Heeney,
H. Kornfeld,
P. R. Donahue,
G. H. Learn,
L. Hood, and J. I. Mullins.
1998.
Evolution of a simian immunodeficiency virus pathogen.
J. Virol.
72:405-414[Abstract/Free Full Text].
|
| 21.
|
Garcia, J. V., and A. D. Miller.
1991.
Serine phosphorylation-independent downregulation of cell-surface CD4 by Nef.
Nature
350:508-511[CrossRef][Medline].
|
| 22.
|
Greenough, T. C.,
J. L. Sullivan, and R. C. Desrosiers.
1999.
Declining CD4 T-cell counts in a person infected with nef-deleted HIV-1.
N. Engl. J. Med.
340:236-237[Free Full Text].
|
| 23.
|
Grzesiek, S.,
A. Bax,
G. M. Clore,
A. M. Gronenborn,
J. S. Hu,
J. Kaufman,
I. Palmer,
S. J. Stahl, and P. T. Wingfield.
1996.
The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase.
Nat. Struct. Biol.
3:340-345[CrossRef][Medline].
|
| 24.
|
Gundlach, B. R.,
S. Reiprich,
S. Sopper,
R. E. Means,
U. Dittmer,
K. Matzrensing,
C. Stahlhennig, and K. Uberla.
1998.
Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines.
J. Virol.
72:7846-7851[Abstract/Free Full Text].
|
| 25.
|
Howe, A. Y. M.,
J. U. Jung, and R. C. Desrosiers.
1998.
Zeta chain of the T-cell receptor interacts with Nef of simian immunodeficiency virus and human immunodeficiency virus type 2.
J. Virol.
72:9827-9834[Abstract/Free Full Text].
|
| 26.
|
Hu, W. S., and H. M. Temin.
1990.
Retroviral recombination and reverse transcription.
Science
250:1227-1233[Abstract/Free Full Text].
|
| 27.
|
Huang, Y.,
L. Zhang, and D. D. Ho.
1995.
Biological characterization of nef in long-term survivors of human immunodeficiency virus type 1 infection.
J. Virol.
69:8142-8146[Abstract].
|
| 28.
|
Hulskotte, E. G.,
A. M. Geretti, and A. D. Osterhaus.
1998.
Towards an HIV-1 vaccine: lessons from studies in macaque models.
Vaccine
16:904-915[CrossRef][Medline].
|
| 29.
|
Ilyinskii, P. O., and R. C. Desrosiers.
1998.
Identification of a sequence element immediately upstream of the polypurine tract that is essential for replication of simian immunodeficiency virus.
EMBO J.
17:3766-3774[CrossRef][Medline].
|
| 30.
|
Jetzt, A. E.,
H. Yu,
G. J. Klarmann,
Y. Ron,
B. D. Preston, and J. P. Dougherty.
2000.
High rate of recombination throughout the human immunodeficiency virus type 1 genome.
J. Virol.
74:1234-1240[Abstract/Free Full Text].
|
| 31.
|
Jin, X.,
D. E. Bauer,
S. E. Tuttleton,
S. Lewin,
A. Gettie,
J. Blanchard,
C. E. Irwin,
J. T. Safrit,
J. Mittler,
L. Weinberger,
L. G. Kostrikis,
L. Q. Zhang,
A. S. Perelson, and D. D. Ho.
1999.
Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques.
J. Exp. Med.
189:991-998[Abstract/Free Full Text].
|
| 32.
|
Kestler, H. W. D.,
D. J. Ringler,
K. Mori,
D. L. Panicali,
P. K. Sehgal,
M. D. Daniel, and R. C. Desrosiers.
1991.
Importance of the nef gene for maintenance of high virus loads and for development of AIDS.
Cell
65:651-662[CrossRef][Medline].
|
| 33.
|
Kirchhoff, F.,
T. C. Greenough,
D. B. Brettler,
J. L. Sullivan, and R. C. Desrosiers.
1995.
Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection.
N. Engl. J. Med.
332:228-232[Free Full Text].
|
| 34.
|
Kirchhoff, F.,
H. W. R. Kestler, and R. C. Desrosiers.
1994.
Upstream U3 sequences in simian immunodeficiency virus are selectively deleted in vivo in the absence of an intact nef gene.
J. Virol.
68:2031-2037[Abstract/Free Full Text].
|
| 35.
|
Kornfeld, H.,
N. Riedel,
G. A. Viglianti,
V. Hirsch, and J. I. Mullins.
1987.
Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses.
Nature
326:610-613[CrossRef][Medline].
|
| 36.
|
Lafont, B. A. P.,
Y. Rivière,
L. Gloeckler,
C. Beyer,
B. Hurtrel,
M. P. Kieny,
A. Kirn, and A. M. Aubertin.
2000.
Implication of the C-terminal domain of Nef protein in the reversion to pathogenicity of attenuated SIVmacBK28-41 in macaques.
Virology
266:286-298[CrossRef][Medline].
|
| 37.
|
Lee, C. H.,
K. Saksela,
U. A. Mirza,
B. T. Chait, and J. Kuriyan.
1996.
Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain.
Cell.
85:931-942[CrossRef][Medline].
|
| 38.
|
Lewis, M. G.,
J. Yalley-Ogunro,
J. J. Greenhouse,
T. P. Brennan,
J. B. Jiang,
T. C. VanCott,
J. C. Lu,
G. A. Eddy, and D. L. Birx.
1999.
Limited protection from a pathogenic chimeric simian-human immunodeficiency virus challenge following immunization with attenuated simian immunodeficiency virus.
J. Virol.
73:1262-1270[Abstract/Free Full Text].
|
| 39.
|
Lohman, B. L.,
M. B. McChesney,
C. J. Miller,
E. McGowan,
S. M. Joye,
K. K. Van Rompay,
E. Reay,
L. Antipa,
N. C. Pedersen, and M. L. Marthas.
1994.
A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge.
J. Virol.
68:7021-7029[Abstract/Free Full Text].
|
| 40.
|
Lole, K. S.,
R. C. Bollinger,
R. S. Paranjape,
D. Gadkari,
S. S. Kulkarni,
N. G. Novak,
R. Ingersoll,
H. W. Sheppard, and S. C. Ray.
1999.
Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.
J. Virol.
73:152-160[Abstract/Free Full Text].
|
| 41.
|
Mariani, R.,
F. Kirchhoff,
T. C. Greenough,
J. L. Sullivan,
R. C. Desrosiers, and J. Skowronski.
1996.
High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection.
J. Virol.
70:7752-7764[Abstract].
|
| 42.
|
Matano, T.,
R. Shibata,
C. Siemon,
M. Connors,
H. C. Lane, and M. A. Martin.
1998.
Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques.
J. Virol.
72:164-169[Abstract/Free Full Text].
|
| 43.
|
McChesney, M. B.,
J. R. Collins,
D. Lu,
X. Lu,
J. Torten,
R. L. Ashley,
M. W. Cloyd, and C. J. Miller.
1998.
Occult systemic infection and persistent simian immunodeficiency virus (SIV)-specific CD4+-T-cell proliferative responses in rhesus macaques that were transiently viremic after intravaginal inoculation of SIV.
J. Virol.
72:10029-10035[Abstract/Free Full Text].
|
| 44.
|
Michael, N. L.,
G. Chang,
L. A. d'Arcy,
C. J. Tseng,
D. L. Birx, and H. W. Sheppard.
1995.
Functional characterization of human immunodeficiency virus type 1 nef genes in patients with divergent rates of disease progression.
J. Virol.
69:6758-6769[Abstract].
|
| 45.
|
Miller, C. J.,
M. B. McChesney,
X. Lu,
P. J. Dailey,
C. Chutkowski,
D. Lu,
P. Brosio,
B. Roberts, and Y. Lu.
1997.
Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239.
J. Virol.
71:1911-1921[Abstract].
|
| 46.
|
Morris, A.,
M. Marsden,
K. Halcrow,
E. S. Hughes,
R. P. Brettle,
J. E. Bell, and P. Simmonds.
1999.
Mosaic structure of the human immunodeficiency virus type 1 genome infecting lymphoid cells and the brain: evidence for frequent in vivo recombination events in the evolution of regional populations.
J. Virol.
73:8720-8731[Abstract/Free Full Text].
|
| 47.
|
Norley, S.,
B. Beer,
D. Binninger-Schinzel,
C. Cosma, and R. Kurth.
1996.
Protection from pathogenic SIVmac challenge following short-term infection with a nef-deficient attenuated virus.
Virology
219:195-205[CrossRef][Medline].
|
| 48.
|
Peeters, M.,
F. Liegeois,
N. Torimiro,
A. Bourgeois,
E. Mpoudi,
L. Vergne,
E. Saman,
E. Delaporte, and S. Saragosti.
1999.
Characterization of a highly replicative intergroup M/O human immunodeficiency virus type 1 recombinant isolated from a Cameroonian patient.
J. Virol.
73:7368-7375[Abstract/Free Full Text].
|
| 49.
|
Petry, H.,
U. Dittmer,
C. Stahl-Hennig,
C. Coulibaly,
B. Makoschey,
D. Fuchs,
H. Wachter,
T. Tolle,
C. Morys-Wortmann, and F. J. Kaup.
1995.
Reactivation of human immunodeficiency virus type 2 in macaques after simian immunodeficiency virus SIVmac superinfection.
J. Virol.
69:1564-1574[Abstract].
|
| 50.
|
Pohlmann, S.,
S. Floss,
P. O. IIyinskii,
T. Stamminger, and F. Kirchhoff.
1998.
Sequences just upstream of the simian immunodeficiency virus core enhancer allow efficient replication in the absence of NF-B and Sp1 binding elements.
J. Virol.
72:5589-5598[Abstract/Free Full Text].
|
| 51.
|
Putkonen, P.,
L. Walther,
Y. J. Zhang,
S. L. Li,
C. Nilsson,
J. Albert,
P. Biberfeld,
R. Thorstensson, and G. Biberfeld.
1995.
Long-term protection against SIV-induced disease in macaques vaccinated with a live attenuated HIV-2 vaccine.
Nat. Med.
1:914-918[CrossRef][Medline].
|
| 52.
|
Regier, D. A., and R. C. Desrosiers.
1990.
The complete nucleotide sequence of a pathogenic molecular clone of simian immunodeficiency virus.
AIDS Res. Hum. Retroviruses
6:1221-1231[Medline].
|
| 53.
|
Robertson, D. L.,
B. H. Hahn, and P. M. Sharp.
1995.
Recombination in AIDS viruses.
J. Mol. Evol.
40:249-259[CrossRef][Medline].
|
| 54.
|
Rud, E. W.,
M. Cranage,
J. Yon,
J. Quirk,
L. Ogilvie,
N. Cook,
S. Webster,
M. Dennis, and B. E. Clarke.
1994.
Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.
J. Gen. Virol.
75:529-543[Abstract/Free Full Text].
|
| 55.
|
Salvi, R.,
A. R. Garbuglia,
A. Dicaro,
S. Pulciani,
F. Montella, and A. Benedetto.
1998.
Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor.
J. Virol.
72:3646-3657[Abstract/Free Full Text].
|
| 56.
|
Sanfridson, A.,
B. R. Cullen, and C. Doyle.
1994.
The simian immunodeficiency virus Nef protein promotes degradation of CD4 in human T cells.
J. Biol. Chem.
269:3917-3920[Abstract/Free Full Text].
|
| 57.
|
Sawai, E. T.,
I. H. Khan,
P. M. Montbriand,
B. M. Peterlin,
C. Cheng-Mayer, and P. A. Luciw.
1996.
Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques.
Curr. Biol.
6:1519-1527[CrossRef][Medline].
|
| 58.
|
Schmitz, J. E.,
M. J. Kuroda,
S. Santra,
V. G. Sasseville,
M. A. Simon,
M. A. Lifton,
P. Racz,
K. Tenner-Racz,
M. Dalesandro,
B. J. Scallon,
J. Ghrayeb,
M. A. Forman,
D. C. Montefiori,
E. P. Rieber,
N. L. Letvin, and K. A. Reimann.
1999.
Control of viremia in simian immunodeficiency virus infection by CD8(+) lymphocytes.
Science
283:857-860[Abstract/Free Full Text].
|
| 59.
|
Schwartz, O.,
V. Marechal,
O. Danos, and J. M. Heard.
1995.
Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.
J. Virol.
69:4053-4059[Abstract].
|
| 60.
|
Schwartz, O.,
V. Marechal,
S. Le Gall,
F. Lemonnier, and J. M. Heard.
1996.
Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.
Nat. Med.
2:338-342[CrossRef][Medline].
|
| 61.
|
Shibata, R.,
C. Siemon,
S. C. Czajak,
R. C. Desrosiers, and M. A. Martin.
1997.
Live, Attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus.
J. Virol.
71:8141-8148[Abstract].
|
| 62.
|
Spouge, J. L.
1992.
Statistical analysis of sparse infection data and its implications for retroviral treatment trials in primates.
Proc. Natl. Acad. Sci. USA
89:7581-7585[Abstract/Free Full Text].
|
| 63.
|
Stahl-Hennig, C.,
U. Dittmer,
T. Nisslein,
H. Petry,
E. Jurkiewicz,
D. Fuchs,
H. Wachter,
K. Matz-Rensing,
E. M. Kuhn,
F. J. Kaup,
E. W. Rud, and G. Hunsmann.
1996.
Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus.
J. Gen. Virol.
77:2969-2981[Abstract/Free Full Text].
|
| 64.
|
Stebbings, R.,
J. Stott,
N. Almond,
R. Hull,
E. Lines,
P. Silvera,
R. Sangster,
T. Corcoran,
J. Rose,
S. Cobbold,
F. Gotch,
A. McMichael, and B. Walker.
1998.
Mechanisms of protection induced by attenuated simian immunodeficiency virus I-lymphocyte depletion does not abrogate protection.
AIDS Res. Hum. Retroviruses
14:1187-1198[Medline].
|
| 65.
|
Switzer, W. M.,
S. Wiktor,
V. Soriano,
A. Silva-Graca,
K. Mansinho,
I. M. Coulibaly,
E. Ekpini,
A. E. Greenberg,
T. M. Folks, and W. Heneine.
1998.
Evidence of Nef truncation in human immunodeficiency virus type 2 infection.
J. Infect. Dis.
177:65-71[Medline].
|
| 66.
|
Takehisa, J.,
M. Osei-Kwasi,
N. K. Ayisi,
O. Hishida,
T. Miura,
T. Igarashi,
J. Brandful,
W. Ampofo,
V. B. Netty,
M. Mensah,
M. Yamashita,
E. Ido, and M. Hayami.
1997.
Phylogenetic analysis of HIV type 2 in Ghana and intrasubtype recombination in HIV type 2.
AIDS Res. Hum. Retroviruses
13:621-623[Medline].
|
| 67.
|
Titti, F.,
L. Sernicola,
A. Geraci,
G. Panzini,
S. Di Fabio,
R. Belli,
F. Monardo,
A. Borsetti,
M. T. Maggiorella,
M. Koanga-Mogtomo,
F. Corrias,
R. Zamarchi,
A. Amadori,
L. Chieco-Bianchi, and P. Verani.
1997.
Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.
J. Gen. Virol.
78:2529-2539[Abstract].
|
| 68.
|
Whatmore, A. M.,
N. Cook,
G. A. Hall,
S. Sharpe,
E. W. Rud, and M. P. Cranage.
1995.
Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence.
J. Virol.
69:5117-5123[Abstract].
|
| 69.
|
Wooley, D. P.,
R. A. Smith,
S. Czajak, and R. C. Desrosiers.
1997.
Direct demonstration of retroviral recombination in a rhesus monkey.
J. Virol.
71:9650-9653[Abstract].
|
| 70.
|
Wyand, M. S.,
K. Manson,
D. C. Montefiori,
J. D. Lifson,
R. P. Johnson, and R. C. Desrosiers.
1999.
Protection by live, attenuated simian immunodeficiency virus against heterologous challenge.
J. Virol.
73:8356-8363[Abstract/Free Full Text].
|
| 71.
|
Wyand, M. S.,
K. H. Manson,
M. Garcia-Moll,
D. Montefiori, and R. C. Desrosiers.
1996.
Vaccine protection by a triple deletion mutant of simian immunodeficiency virus.
J. Virol.
70:3724-33[Abstract].
|
| 72.
|
Wyand, M. S.,
K. H. Manson,
A. A. Lackner, and R. C. Desrosiers.
1997.
Resistance of neonatal monkeys to live attenuated vaccine strains of simian immunodeficiency virus.
Nat. Med.
3:32-6[CrossRef][Medline].
|
| 73.
|
Zhang, J., and H. M. Temin.
1994.
Retrovirus recombination depends on the length of sequence identity and is not error prone.
J. Virol.
68:2409-2414[Abstract/Free Full Text].
|
Journal of Virology, February 2001, p. 1507-1515, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1507-1515.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
