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Journal of Virology, June 1999, p. 4952-4961, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Highly Attenuated Vaccine Strains of Simian Immunodeficiency
Virus Protect against Vaginal Challenge: Inverse Relationship of
Degree of Protection with Level of Attenuation
R. Paul
Johnson,1
Jeffrey D.
Lifson,2
Susan C.
Czajak,1
Kelly Stefano
Cole,3
Kelledy H.
Manson,4
Rhona
Glickman,1
Janet
Yang,1
David C.
Montefiori,5
Ronald
Montelaro,3
Michael S.
Wyand,4 and
Ronald C.
Desrosiers1,*
New England Regional Primate Research Center,
Harvard Medical School, Southborough, Massachusetts
01772-91021; NCI-Frederick Cancer
Research and Development Center, Frederick, Maryland
217012; Department of Molecular Genetics
and Biochemistry, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 152613;
Primedica, Worcester, Massachusetts
016084; and Department of Surgery,
Duke University Medical Center, Durham, North Carolina
277105
Received 2 December 1998/Accepted 26 February 1999
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ABSTRACT |
Three different deletion mutants of simian immunodeficiency virus
(SIV) that vary in their levels of attenuation were tested for the
ability to protect against mucosal challenge with pathogenic SIV. Four
female rhesus monkeys were vaccinated by intravenous inoculation with
SIVmac239
3, four with SIVmac2393X, and four with SIVmac2394.
These three vaccine strains exhibit increasing levels of attenuation:
3 < 3X <4. The vaccinated monkeys were challenged by
vaginal exposure to uncloned, pathogenic SIVmac251 at 61 weeks after
the time of vaccination. On the basis of viral RNA loads in plasma,
cell-associated virus loads in peripheral blood, and CD4 cell counts,
strong protective effects were observed in all three groups of
vaccinated monkeys. However, the degree of protection correlated
inversely with the level of attenuation; the least-attenuated strain,
SIVmac2393, gave the greatest protection. One monkey in the 3X
group and two in the 4 group clearly became superinfected by the
challenge virus, but these animals had levels of SIV RNA in plasma that
were considerably lower than those of naive animals that were
challenged in parallel. Protection against vaginal challenge appears
easier to achieve than protection against intravenous challenge, since
four other SIVmac2394-vaccinated monkeys showed no protection when
challenged intravenously with a much lower inoculum of the same
challenge virus stock. Protection against vaginal challenge in the
4-vaccinated group occurred in the absence of detectable serum
neutralizing activities and appeared to be associated with the
development of an early SIV-specific cytotoxic-T-lymphocyte response.
Our results demonstrate that mucosal protection can be achieved by
systemic immunization with the highly attenuated SIVmac2394 more
than 1 year prior to the time of challenge.
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INTRODUCTION |
Live, attenuated simian
immunodeficiency virus (SIV) deletion mutants have strongly protected
rhesus monkeys against challenge by pathogenic strains of the virus
(1, 4, 6, 34). Better knowledge of the features of this
protection will be needed to move the live, attenuated vaccine approach
for AIDS forward. At the very least, better understanding of the
protection will aid in designing other vaccine approaches that can
mimic it.
By analogy to other viral systems (11, 12), we might expect
some viral strains to be lacking in safety because they are not
attenuated enough and others to be lacking in protective efficacy because they are too attenuated. Thus, an important consideration for
live, attenuated AIDS vaccines is the balance between safety and
efficacy. A wide range of attenuation has been achieved in SIV by varying the number and location of deletion mutations
(9). However, comparative analysis of the protective
capacities of these different vaccine strains has not been undertaken.
Such systematic comparisons may also provide clues to the immune
responses associated with protection by live, attenuated SIV.
Although the majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide occur via mucosal transmission, most AIDS
vaccine trials in monkeys have analyzed the abilities of different
vaccines to protect against intravenous rather than mucosal challenge
(30). Most studies of live, attenuated SIV deletion mutants have similarly examined the ability to protect against
intravenous challenge with pathogenic SIV (1, 4, 6,
34), although at least one study has reported that systemic vaccination with a nef-defective strain of SIV
protected against rectal exposure (5). There is a compelling
need to provide more information on the ability of AIDS vaccines to
provide protection against mucosal challenge.
Three different attenuated derivatives of SIVmac239
were compared for the ability to protect against vaginal
challenge by an uncloned, pathogenic, slightly heterologous strain,
SIVmac251. Protection appeared to vary inversely with the
degree of attenuation. However, even a highly attenuated derivative
lacking nef, vpr, vpx, and upstream
sequences in U3 (SIVmac2394) induced a reasonable level of
protection against vaginal challenge.
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MATERIALS AND METHODS |
Virus stocks, immunization, and challenge.
The vaccine
strains SIVmac2393, -3X, and -4 were produced by
transfection of cloned DNA into CEMx174 cells, and virus was harvested
from the cell-free supernatant at or near the peak of virus-induced
cytopathic effect 7 to 11 days later. The actual stocks used for these
experiments have been described (9). SIVmac2393
is missing unique nef, vpr, and nef
sequences that overlap U3 (US); SIVmac2393X is missing
nef, vpx, and US sequences; SIVmac2394 is missing nef, vpr,
vpx, and US (9). Vaccine virus containing 100 ng
of p27 was administered to each animal by intravenous inoculation.
Intravenous challenge with uncloned, early-passage SIVmac251
was done with 10 rhesus monkey infectious doses (virus containing 0.032 ng of p27). The preparation, titration, and use of this challenge stock
has been described previously (6, 18, 34). This same stock
was used for vaginal challenge. Undiluted virus (0.5 ml) containing 48 ng of p27 was placed in the vaginal cavity atraumatically with the
animal in a horizontal or slightly inverted position.
Virus load measurements.
The number of infectious cells in
peripheral blood mononuclear cells (PBMC), i.e., the cell-associated
virus load, was quantitated as previously described (9).
Quantitation of viral RNA levels in plasma by real-time reverse
transcriptase PCR has also been described (31).
Other measurements.
CD4 cell numbers were quantitated by
flow cytometry with a whole-blood lysis technique that we have used
previously (34). Procedures for amplifying across the
nef gene by PCR for the analysis of wild-type (WT) versus
vaccine sequences have been described (34). SIV
was purified with the use of column chromatography and used to
coat enzyme-linked immunosorbent assay (ELISA) plates as described
previously (7). The presence of antibodies to SIV
was detected with alkaline phosphatase-conjugated goat anti-human immunoglobulin G, which we have also used previously (9,
34). Procedures for the measurement of neutralization of
SIV were performed as described previously (17,
34).
Measurement of viral envelope glycoprotein-specific
antibody endpoint titer, conformational dependence, and avidity by ConA
ELISA.
Serum samples from macaques infected with
SIVmac239 deletion mutants (3, 3X, and 4) were
analyzed for their reactivity to SIVsmB7 (15)
viral envelope glycoproteins in a concanavalin A (ConA)
ELISA as previously described (3). Endpoint titers to viral
envelope glycoproteins are reported as the last serial twofold dilution whose optical density was twice that of normal monkey
serum or an optical density of 0.1, whichever value was greater, and
all endpoint titer values represent at least two independent
experiments. Measurements of conformational dependence were calculated
from the ratios of serum antibody reactivities to native envelope
glycoprotein substrates versus those to denatured substrates. Thus, the conformation ratio is a direct measure of the
conformational dependence of a particular antibody sample (i.e., the
larger the conformation ratio above 1.0, the greater the requirement
for native envelope glycoprotein structure, while conformation ratios below 1.0 reflect predominant specificity for
linear envelope determinants). Viral envelope
glycoprotein-specific antibody avidities were determined by
measuring the resistance of serum antibody-envelope
glycoprotein complexes to treatment with 8 M urea in the
ConA ELISA. The avidity index was then calculated from the ratio of the
absorbance value obtained with urea treatment to that observed with
phosphate-buffered saline treatment multiplied by 100%. All
conformation ratios and avidity index values represent at least three
independent experiments with several different serum dilutions within
the linear range to ensure that the variation in actual values was
within 10%.
Assay of SIV-specific CTL activity.
PBMC were
isolated from fresh heparinized blood by centrifugation over a
Ficoll-sodium diatrizoate (Ficoll 1077; Sigma, St. Louis, Mo.) gradient
and suspended at 2 × 106/ml in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM
L-glutamine, 50 IU of penicillin/ml, and 50 µg of
streptomycin/ml (R10). Antigen-specific stimulation of PBMC was
carried out as described previously (14) with autologous B
lymphoblastoid cell lines (B-LCL) infected with a recombinant vaccinia
virus (vAbt388; provided by D. Panicali, Therion Biologics, Cambridge,
Mass.) containing the SIVmac251 gag and
pol genes and the SIVmac239 env gene.
After an overnight incubation, vaccinia virus-infected B-LCL were
inactivated with UV and psoralen and cultured with PBMC at a
responder-to-stimulator ratio of 10:1 in R10 medium. Recombinant
interleukin-2 (provided by Maurice Gately, Hoffman LaRoche) was added
to a final concentration of 10 to 20 U/ml on day 4 of culture.
Cytotoxic-T-lymphocyte (CTL) assays were performed 10 to 14 days after
stimulation. The target cells consisted of autologous B-LCL infected
with recombinant vaccinia viruses expressing SIV proteins.
The recombinant vaccinia viruses used to infect the target cells
included vAbt252 (encoding the SIVmac251
p55gag and protease proteins; Therion), rVV-239
(encoding the SIVmac239 envelope; provided by M. Mulligan
[28]), and the control vaccinia virus NYCBH. B-LCL
were infected overnight with a multiplicity of infection of 5 to 10 PFU/cell and then labeled with chromium 51 (DuPont NEN, Wilmington,
Del.) at 100 µCi per 106 cells. Target and effector cells
(104/well) were dispensed in duplicate for each
effector/target (E/T) ratio into 96-well U-bottom plates (Costar). To
decrease background lysis, cold targets consisting of unlabeled
autologous B-LCL infected with the control vaccinia virus NYCBH were
used at a cold target/hot target ratio of 15:1. Chromium release was
assayed after a 5-h incubation at 37°C in a 5% CO2
incubator. The plates were spun at 1,000 rpm for 10 min at 4°C, after
which 30 µl of supernatant was harvested from each well into wells of
a LumaPlate-96 (Packard) and allowed to dry overnight. Emitted
radioactivity was measured in a 1450 MicroBeta Plus liquid
scintillation counter (Wallac, Turku, Finland). Spontaneous release was
measured from wells containing only target cells and medium. Maximum
release was measured from wells containing target cells and 0.1%
Triton X-100 (Sigma). The percent cytotoxicity was calculated as
follows: [(test release 
spontaneous release)/(maximum
release spontaneous release)] × 100. SIV-specific
cytotoxicity was calculated by subtracting the lysis of NYCBH-infected
target cells from that obtained with targets expressing SIV
proteins. Spontaneous release of target cells was <25% in all assays.
E/T ratios for which background lysis of control targets exceeded 20%
were excluded from analysis. Based on examination of 10 naive control
animals not infected with SIV, SIV-specific lysis
of greater than 5% seen at more than one E/T ratio was interpreted as significant.
Statistical analysis. Mann-Whitney tests and analysis of
variance (ANOVA) were performed using StatView (Abacus Concepts, Berkeley, Calif.).
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RESULTS |
Vaccine phase.
The groups of four female rhesus monkeys were
vaccinated by intravenous inoculation with normalized amounts of
vaccine virus containing 100 ng of p27 antigen. One group received
SIVmac2393, one received SIVmac2393x, and one
received SIVmac2394. These strains have been described
(9). The cell-associated virus loads, plasma viral RNA
loads, and antibody responses following vaccination of these twelve
animals have recently been described (9).
Virus loads following vaginal challenge.
All 12 vaccinated
monkeys and four unvaccinated controls were exposed to
SIVmac251 challenge virus vaginally at 61 weeks after the
time of vaccination. This is the same stock of virus that has been used
for intravenous challenge in our previous studies (6, 8,
34). All four of the control animals became persistently infected
on the basis of persistent virus recovery from PBMC (Fig. 1) and persisting high levels of viral
RNA in plasma (Fig. 2). Both measures of
virus load in these control animals were similar to what we have seen
previously in animals infected with the same virus by the intravenous
route (34). In addition to these four controls, two other
rhesus monkeys that were passively administered antibody as part of
other experiments also became infected when exposed vaginally at the
same time with the same stock (data not shown). Two of two additional
animals inoculated vaginally with a 1:5 dilution of SIV were
also infected (data not shown). In all, we have exposed a total of
eight unvaccinated female rhesus monkeys by the same route with the
same stock used for these experiments, or a 1:5 dilution of it, and all
eight have become infected.
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FIG. 1.
Cell-associated virus loads in monkeys (Mm) challenged
with WT SIVmac251 by the vaginal route. The numbers of
infectious cells in PBMC were quantitated as described in Materials
and Methods. Code for PBMC load: 0, virus was not recovered even
when 106 PBMC were used; 1, virus was recovered with an
average of 106 but not fewer PBMC; 2, 333,333 PBMC;
3, 111,111 PBMC; 4, 37,037 PBMC; 5, 12,345 PBMC; 6, 4,115 PBMC; 7, 1,371 PBMC; 8, 457 PBMC.
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FIG. 2.
Loads measured as amounts of viral RNA in plasma in
monkeys (Mm) challenged with WT SIVmac251 by the vaginal
route. The dashed lines indicate threefold sensitivity of the assay,
i.e., 300 copy equivalents (Eq) per ml.
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Vaccine virus was not recovered from PBMC of 11 of the 12 vaccinated animals on the day of challenge, or immediately prior
to it,
even when 10
6 PBMC were used (Fig.
1). Viral RNA in
plasma was also below the
threshold of detection (<300 copy
equivalents per ml) in all 12
animals prior to challenge. This is
consistent with the attenuated
nature of these vaccine strains
described previously (
9).
In contrast to the control animals, all four of the
3-vaccinated
animals exhibited little or no SIV recovery from PBMC for
a prolonged period after challenge even when 10
6 PBMC
were used (Fig.
1). Viral RNA in plasma also remained around
or below
the limit of detection in the four
3-vaccinated monkeys
(Fig.
2).
One of the four monkeys vaccinated with
3x (430-93)
exhibited a
sharp spike in cell-associated virus loads at 2 weeks
postchallenge
(Fig.
1). This corresponded to a similar spike in
viral RNA in the
plasma of the same animal (Fig.
2). Viral RNA
in plasma in this animal
persisted at measurable levels at all
time points examined over the
course of more than 60 weeks (Fig.
2). However, the persisting level of
viral RNA was about 100-fold
lower in this animal than in the controls
in this experiment (Fig.
2) and in similar controls used previously
(data not shown). One
other animal in the
3x challenge group
(466-93) exhibited measurable
levels of viral RNA at some time points
postchallenge, although
genetic analysis of viral DNA in PBMC
revealed only
nef sequences
(see below) (Fig.
2). The
other two monkeys in the
3x challenge
group were completely
protected as assessed by viral cultures
and viral RNA. Two of the four
4-vaccinated monkeys became virus
recovery positive postchallenge
(Fig.
1), and both of these monkeys
developed measurable levels of
persisting viral RNA postchallenge
(Fig.
2). The levels of viral RNA in
these animals 8 to 16 weeks
postchallenge were about 100-fold lower
than the levels observed
in the controls for this experiment (Fig.
2)
and in similar controls
in other experiments (data not shown). The
other two
4-vaccinated
monkeys appeared to be solidly protected as
measured by the virus
load
criteria.
To differentiate vaccine strain SIV from the challenge virus
SIVmac251, DNA was prepared from PBMC of all challenged
animals
at weeks 2 and 24 postchallenge and used for genetic analysis
by PCR. Primers that span the
nef gene were used in such a
way
that DNA of WT challenge virus and DNA of vaccine strains from
which
nef was deleted yielded different-sized fragments
following
amplification (
15,
34). The results of these
genetic analyses
agreed well with the results of all other analyses.
PCR analysis
of PBMC from animals 430-93 in the
3X group and
445-93 and 465-93
in the
4 group yielded a PCR product of WT size at
both 2 and
24 weeks postchallenge (Table
1). All of the other animals were
either
negative for amplification of viral DNA or yielded PCR
products
consistent with the size of the product of the vaccine
strain with the
nef deletion at both time points (Table
1).
Intravenous challenge of animals vaccinated with
SIVmac2394.
Four other monkeys that had been
vaccinated with SIVmac2394 were challenged in separate
experiments by the intravenous route. The same stock of
SIVmac251 that was used in the vaginal challenge experiments
was also used for the intravenous challenges. The titers of this stock
have been determined previously by the intravenous route in rhesus
monkeys (18). Ten rhesus monkey infectious doses (intravenous) were used for the intravenous challenge. The challenge was performed 75 weeks after the time of vaccination. Virus load measurements postchallenge demonstrated no protective effects of the
4 vaccination (Fig. 3). A fifth monkey
that had been vaccinated with SIVmac2394 33 weeks
previously was similarly challenged, and again there were no protective
effects (data not shown).
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FIG. 3.
Cell-associated virus loads in 4-vaccinated monkeys
challenged intravenously. Weeks indicate weeks postvaccination with
SIVmac2394. The animals were challenged at week 75 intravenously with SIVmac251. The numbers of infectious cells
in PBMC are indicated on the y axis. The code for
PBMC load is the same as that in the legend to Fig. 1. L41 is an
unvaccinated control challenged in parallel.
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CD4+-T-cell counts and anamnestic antibody
responses.
Changes in the percentage and absolute number of
CD4+ T cells were quite consistent with the virus load
measurements. All 12 of the vaccinated monkeys in the vaginal-challenge
group exhibited percentages of CD4+ T cells in the normal
range throughout the vaccine phase and at the time of challenge (Fig.
4). All four of the 3-vaccinated animals maintained stable percentages of CD4+ T cells for
greater than 1 year of follow-up postchallenge (Fig. 4). However,
430-93 in the 3X group and 445-93 and 465-93 in the 4 group
exhibited significant declines in the CD4+-cell population
following vaginal challenge (Fig. 4). These same animals also developed
consistently measurable viral loads postchallenge (Fig. 1 and 2). An
additional 3x-vaccinated animal, 466-93, showed a rising and then
falling percentage of CD4+ T cells (Fig. 4). Two of the
unvaccinated control monkeys that were challenged (168-95 and 387-95)
exhibited declining percentages of CD4+ T cells up until
the time of their deaths from AIDS at 29 and 30 weeks postchallenge
(Fig. 4). 315-95, an unvaccinated monkey, and 445-93 and 465-93, unprotected monkeys in the 4 challenge group, also died from AIDS at
69, 39, and 82 weeks postchallenge, respectively. The other 10 vaccinated monkeys in this study were still alive at 86 weeks
postchallenge. The four 4-vaccinated monkeys that were challenged
intravenously (Fig. 3) all exhibited declines in CD4+-cell
counts (Fig. 5), and three of the four
died or had to be euthanized due to AIDS. For all animals, analysis of
the absolute number of CD4+ T cells demonstrated changes
similar to those seen with analysis of the percentage of
CD4+ T cells (data not shown).
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FIG. 4.
Percentages of CD4+ T lymphocytes in monkeys
(Mm) challenged with SIVmac251 by the vaginal route. The
results are expressed as the percentage of cells in PBMC that were
CD4+ T lymphocytes.
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FIG. 5.
Percentages of CD4+ T lymphocytes in
4-vaccinated monkeys challenged intravenously. The animals were
challenged intravenously with SIVmac251 at 75 weeks. L41 is
the unvaccinated control challenged in parallel. The results are
expressed as the percentage of cells in PBMC that were
CD4+ T lymphocytes.
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We also used anti-SIV antibody levels to look for anamnestic
antibody responses. Animals 445-93 and 465-93 in the
4 group
and
430-93 in the
3X group had clear, strong anamnestic antibody
responses postchallenge (Fig.
6),
consistent with the other measurements
described above. None of the
other vaccinated animals that were
challenged vaginally showed any
clear evidence of anamnestic responses
(Fig.
6). All four of the
4-vaccinated monkeys that were challenged
intravenously also
exhibited anamnestic responses (data not shown).
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FIG. 6.
Anamnestic antibody responses in monkeys (Mm) challenged
vaginally. Plasma from monkeys obtained at the indicated weeks were
reacted with purified, lysed SIV, using 1:200 dilutions of
plasma and 1:100 dilutions of conjugate. Week 0 is the time of
challenge.
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Analysis of SIV-specific antibody responses.
In an
effort to better understand which immune responses might be involved in
mediating protection induced by these attenuated SIV strains,
we attempted to correlate several measurements of SIV-specific antibodies and neutralizing antibody titers on
the day of challenge with protection. SIV-specific antibody
responses were evaluated with respect to endpoint dilution titer,
conformational ratio, and avidity index. Previous studies of these
parameters in vaccinated animals have shown that monkeys protected by
live, attenuated SIV vaccines often had antibody responses
characterized by a relatively high avidity and lower conformation
dependence, characteristics that have been associated with maturation
of the humoral immune response to SIV (3).
Plasma taken on the day of challenge, just prior to inoculation of the
challenge virus, was used to measure anti-SIV antibody
levels
by using ELISA plates coated with antigens from lysed,
purified
virions. Using a similar assay, we previously showed
that these same
3-,
3X-, and
4-vaccinated animals exhibited
antibody responses
in the initial weeks and months after vaccination
that varied according
to the vaccine strain:
3 >
3X >
4 (
9).
Plasma taken on the day of challenge 61 weeks after the time of
vaccination exhibited this same trend. Among the
4-vaccinated
animals, the two animals that had no detectable replication of
challenge virus (394-93 and 435-93) had the lowest levels of
SIV-specific
antibodies (Fig.
7). In fact, monkey 394-93 in the
4
group, which
originally showed marginal antibody levels around the
borderline
of detection (
9), also showed questionable
reactivity with
plasma taken on the day of challenge (Fig.
7).
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FIG. 7.
Antibody titers to SIV on the day of vaginal
challenge. Plasma obtained from monkeys on the day of, and just prior
to, vaginal challenge were reacted with purified, lysed SIV,
using serial fourfold dilutions starting at 1:20.
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We also examined SIV-specific envelope responses by using a
heterologous envelope from the SIV/B7 molecular clone, which
was
derived from SIVsmmH3 (Fig.
8). Again, the same trend was observed:
SIV envelope-specific titers decreased with increasing
attenuation
of the vaccine strain (Fig.
8). Among the
4-vaccinated
group,
the protected animals had the lowest endpoint titers.
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FIG. 8.
Analysis of envelope-specific anti-SIV
antibody responses on the day of vaginal challenge. Serum samples were
analyzed for reactivity to SIVsmB7 viral envelope
glycoproteins in a ConA ELISA as previously described
(3). Measurements of conformational dependence were
calculated from the ratios of serum antibody reactivities to native
versus denatured envelope glycoprotein substrates.
Measurements of viral envelope glycoprotein-specific
antibody avidity were determined by measuring the resistance of serum
antibody-envelope glycoprotein complexes to mild treatment
with 8 M urea in the ConA ELISA. Unprotected animals (430-93, 445-93, and 465-93) are represented by open symbols; protected animals are
represented by solid symbols.
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SIV-specific antibodies were also characterized with respect
to their conformation ratios and avidity indexes (
3). The
conformation ratio was determined by measuring the serum reactivity
to
native viral glycoproteins compared to that with denatured
viral glycoproteins by ELISA. The avidity index was
determined
by measuring the resistance of serum antibody envelope
glycoprotein
immune complexes to disruption by treatment
with 8 M urea. By
ANOVA,
4-vaccinated animals had significantly
lower avidity indices
(
P = 0.02) and a trend toward
lower endpoint titers and higher
conformational ratios. In general,
animals that were protected
exhibited higher antibody avidity indices
and lower conformational
ratios whereas unprotected animals tended to
have lower avidity
indices and higher conformational ratios (Fig.
8).
Using a Mann-Whitney
test, correlation of protection with avidity index
and with conformational
ratio reached statistical significance
(
P = 0.03 for each). However,
the trend of increased
protection with higher avidity index and
lower conformational ratio
appeared to be largely related to the
degree of attenuation of the
vaccine strain rather than protection
per se, since there was no clear
distinction among the
4-vaccinated
animals in either conformation
ratio or avidity index between
protected and unprotected
animals.
We also measured the ability to neutralize viral infectivity by using a
laboratory-passaged SIVmac251 (251
L) and a
primary
stock of SIVmac251 (251
p). As has been
noted previously, (
17,
22), the laboratory-passaged stock
was more easily neutralized
(Table
2).
Plasma taken on the day of challenge was used for
these measurements.
Six of the eight monkeys vaccinated with SIV
3
and -
3X
showed activity in plasma capable of neutralizing the
laboratory-passaged stock. None of the plasma from the
4-vaccinated
animals, including the two solidly protected animals, had neutralizing
activity to the laboratory-passaged stock (Table
2). Plasma with
the
highest levels of neutralizing activity against the laboratory-passaged
virus had low levels of neutralizing activity against the primary
stock, with titers in the range of 1:10 to 1:24.
Analysis of SIV-specific CTL responses prechallenge.
We also attempted to correlate SIV-specific CTL responses
with the outcome of challenge. CTL responses against SIV Gag
and envelope were prospectively evaluated during the vaccine phase with
fresh PBMC stimulated with autologous B-LCL infected with recombinant vaccinia viruses expressing SIV proteins, as
described previously (14). CTL activity was then assessed at
multiple E/T ratios against autologous B-LCL expressing either
SIV Gag or SIV envelope. As previously reported,
all four animals vaccinated with SIVmac2393 developed
SIV-specific CTL activity within 4 weeks after infection
(14). Serial evaluation of SIV-specific CTL
activity in these animals at later time points revealed several different patterns (Fig. 9A). For two of
the animals (353-93 and 368-93), relatively stable levels of
SIV-specific CTL activity were observed to 61 weeks of
infection. In one animal (330-93), a late increase in
SIV-specific CTL activity was observed at 52 weeks and
confirmed at 59 weeks, whereas in the other animal (419-93) CTL
activity to the target antigens peaked at 8 weeks and decreased thereafter. The levels of SIV-specific CTL activity in these
SIV3-vaccinated animals were generally similar to those
reported in our earlier publication (14).
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FIG. 9.
Prospective analysis of SIV-specific CTL
responses in vaccinated monkeys prior to vaginal challenge. CTL
activity following antigen-specific stimulation was examined at
multiple E/T ratios, and representative data are shown for an E/T of
40:1 for animals vaccinated with SIVmac2393 (A),
SIVmac2393X (B), and SIVmac2394 (C).
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Among the macaques vaccinated with SIVmac239
3x, three of
four animals had developed SIV-specific CTL responses at some
time
point by 20 weeks after infection, although
SIV-specific CTL activity
in these animals was more
consistently observed after 38 weeks
of infection (Fig.
9B). One animal
(430-93), the only unprotected
3x-infected animal, never developed
significant SIV-specific
CTL activity at any time point
examined prior to
challenge.
Two of the 4 animals vaccinated with SIVmac239
4 developed
SIV-specific CTL responses against Gag or envelope in the
first
8 weeks of infection (Fig.
9C). One of these animals (394-93)
had
one of the highest observed CTL responses of any animal studied
in this
vaccine trial. This same animal did not have detectable
viral RNA at
any time and had only marginally detectable levels
of virus binding
antibodies (
9) (Fig.
7). Both of these early
4-vaccinated
CTL responders were solidly protected against vaginal
challenge (Table
2). The other two SIVmac239
4-vaccinated animals,
which
were not completely protected, developed SIV-specific CTL
responses of greater than 10% specific lysis only after 38 to
52 weeks
of
infection.
| |
DISCUSSION |
Our results clearly demonstrate that the majority of
vaccinated monkeys were solidly protected against vaginal
SIVmac251 challenge on the basis of virus load measurements,
stable antibody levels, lack of any declines in CD4+-T-cell
numbers, and genetic analysis of viral sequences in PBMC. We cannot
be certain that there was indeed "sterilizing immunity" in each of
these cases or whether some animals might develop increasing virus
loads and disease progression at a later date. However, our prior
experience with monkeys vaccinated with live, attenuated SIV
with this degree of protection is that such monkeys have remained solidly protected for as long as we have followed them, as long as 6 3/4 years (references 6 and 34
and results not shown). In contrast, 430-93 in the 3X group and
445-93 and 465-93 in the 4 group clearly became superinfected
following vaginal SIV251 challenge and developed virus loads
in the readily measurable range. While the persisting virus loads in
these three animals that were not solidly protected were on average
considerably lower than those in the control, unvaccinated group, our
previous experience with such animals is that they will progress toward
AIDS, but at a slower rate (reference 34 and results
not shown).
Our data indicate that it is easier for live, attenuated SIV
to protect against vaginal mucosal exposure than against intravenous challenge. None of the four 4-vaccinated monkeys that were
challenged intravenously following a similar period of vaccination
showed any level of protection at all, while protective effects were observed in all four of the 4-vaccinated monkeys that were
challenged vaginally: two with solid protection and two with lowering
of viral loads. It is important to note that the same stock of
SIVmac251 was used for both the intravenous and vaginal
challenges. Thus, differences in outcome cannot be ascribed to
differences in the stocks used. It also appears unlikely that
differences in the outcome of challenge reflect differences in the
infectious dose of virus used for each of these routes. The
vaginal-challenge dose used for these experiments contained 1,500 times
more virus than was used for the intravenous challenge (48 ng of p27
antigen vaginally compared with 32 pg intravenously). In vivo
titrations have demonstrated that the 32-pg intravenous-challenge dose
contains 10 monkey infectious doses by this route (18).
Although the vaginal dose used for these experiments has not been
formally titrated, the fact that two of two animals challenged with a
1:5 dilution of this stock became infected suggests that this stock contains at least 5 monkey infectious doses. Thus, the dose of SIVmac251 used for vaginal challenge was likely to contain a
level of animal infectious units of SIV at least comparable
to that present in the intravenous challenge. Using recombinant
poxvirus vaccines, Benson et al. have also observed better protection
against mucosal challenge exposure than against intravenous challenge (2).
These results provide clear evidence for the ability of systemic
immunization with a live, attenuated SIV to provide
protection against mucosal challenge. Systemic immunization with
nonreplicating antigens is relatively inefficient at inducing local
immune responses at mucosal sites, and effective induction of mucosal
immune responses generally requires administration of antigens at the
mucosal surface (23). This feature has led to the suggestion
that effective protection against mucosal exposure to SIV and
HIV may require mucosal immunization (21, 25). Several
previous attempts to induce protection against vaginal infection with
pathogenic SIV have been unsuccessful, including immunization
regimens employing live, attenuated SIV (20) or
targeted lymph node immunization (21). Our present results,
along with the previous demonstration that systemic immunization with a
nef-defective SIV strain can protect against
rectal challenge (5), provide clear documentation for
protection against mucosal challenge following a simple systemic immunization. The immunologic mechanisms responsible for mediating this
protection are not understood and may involve immunity at multiple
sites, including the mucosal surface, submucosa, or regional or
draining lymph nodes. The efficacy of live, attenuated SIV strains in this regard may in part be due to their ability to replicate
significantly at mucosal sites during the early weeks after vaccination
(33). Thus, highly attenuated SIV vaccine strains
may create sufficient immunity to limit challenge virus replication at
the mucosal site or limit its subsequent spread to systemic sites.
Evidence that protection is indeed immune mediated has been summarized
recently (13).
Our results also illustrate the relationship of the level of
attenuation of the vaccine strain with the degree of protection. We
previously reported solid, long-term protection of four monkeys that
had previously been vaccinated with SIV239nef and
subsequently challenged intravenously (6). Recently, Connor
et al. (4) reported strong protection against intravenous
challenge by SIVmac251 in eight monkeys that had been
vaccinated with SIV239nef 15 or more weeks previously.
Similarly, Wyand et al. (34) obtained solid protection in
four of four monkeys that had been persistently infected with
SIV2393 for 79 weeks when they were challenged intravenously with SIV251. In contrast, there were no
protective effects in the present study when four monkeys that were
vaccinated with SIV2394 were challenged
intravenously with SIVmac251 79 weeks after vaccination. It
is important to note that the same challenge stock of
SIVmac251 at the same dose was used for these various
studies, so differences in outcome cannot be ascribed to differences in
the challenge stock used. Similarly, in the vaginal challenges
described in this report, decreasing protection was observed with
increasing levels of attenuation in the vaccine strain, going from 3
to 3X to 4. Thus, the degree of protection appears to correlate
inversely with the level of attenuation of the vaccine strain. Lohman
et al. have also noted an inverse relation of protection with level of
attenuation for the highly attenuated cloned virus SIVmac1A11
and less attenuated derivatives obtained by recombination with
SIVmac239 (19). In considering the live, attenuated vaccine approach for use in people, there will clearly be a
need to balance the desire to attenuate the virus as much as possible
to help ensure safety with the desire to make the vaccine as effective
as possible.
Although infection with increasingly attenuated SIV strains
clearly decreased the strength of the SIV-specific humoral
response (9) (Fig. 7 and Table 2), we did not observe such a
clear relationship for the strength of the CTL response. Although early SIV-specific CTL responses (by 8 weeks of vaccination) were
observed less frequently in animals immunized with
SIVmac2394 than in those immunized with
SIVmac2393, the SIV-specific CTL responses in
those 4 animals that developed early CTL activity were as strong or
stronger than those in animals immunized with 3 or 3X. The effect
of attenuation on the magnitude of immune responses induced by live,
attenuated SIV strains may therefore differ between humoral
and cellular immune responses.
We are encouraged that a strain as attenuated as SIV2394
can still give reasonable levels of protection against natural exposure to a virulent SIV more than 1 year after the time of
vaccination. Although only two of four animals were solidly protected,
the other two animals exhibited virus load reductions compared to unvaccinated controls. It may be possible in future experiments to get protection with even more attenuated strains by the inclusion of
specific types of booster immunizations.
Our studies do not allow unambiguous assignment of immune parameters
that are associated with protection. Factors that may contribute to
this inability include the relatively small number of unprotected
animals, the limited quantitative capabilities of bulk CTL assays,
failure to measure immune responses that are the relevant ones, or
failure to measure them in an appropriate fashion. Alternatively,
protective immunity against primate lentiviruses may result from the
sum effect of multiple different immune responses, each of which may
play some additive role in resistance to infection. Analysis of the
protected 4-vaccinated animals does suggest that protection against
mucosal infection can be achieved in the absence of detectable
neutralizing antibodies in plasma and, in one case, even with
ELISA-binding antibodies at the borderline of detection. Similar
conclusions have been reached by other investigators examining immunization or challenge with chimeric SIV strains that
express the HIV-1 envelope (5, 24). Further analysis of
larger cohorts of animals vaccinated with highly attenuated
SIV strains, coupled with more quantitative assessments of
cellular immune responses, such as those derived from the use of major
histocompatibility complex tetramers (10, 32) should allow
more detailed evidence for immune correlates of protection by live,
attenuated SIV.
| |
ACKNOWLEDGMENTS |
We thank María García-Moll of Bio-Molecular
Technology, Inc., for the genetic analyses. We also thank Dong-Ling
Xia, Allan McPhee, and Theresa Wiltrout for technical assistance,
MaryAnn DeMaria and Michael Rosenzweig for flow cytometric analysis,
Dennis Panicali and Mark Mulligan for providing recombinant vaccinia viruses, Maurice Gately for providing recombinant IL-2, and Joanne Newton and Jane FitzPatrick for manuscript preparation. We also thank
K. Mansfield, P. Sehgal, E. Roberts, and the Division
of Primate Resources for animal care, blood sampling, and
clinical care.
This work has been supported by PHS grants AI35365, AI43044 (R.C.D. and
R.P.J.) AI43075 (R.M.), AI07487 (K.S.C.), and RR 00168 and with federal
funds from the National Cancer Institute, National Institutes of
Health, under contract N01-CO-56000.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: New England
Regional Primate Research Center, Harvard Medical School, One Pine Hill Dr., P.O. Box 9102, Southborough, MA 01772-9102. Phone: (508) 624-8042. Fax: (508) 624-8190. E-mail:
ronald_desrosiers{at}hms.harvard.edu.
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(2005). Simian Human Immunodeficiency Virus-Associated Pneumonia Correlates with Increased Expression of MCP-1, CXCL10, and Viral RNA in the Lungs of Rhesus Macaques. Am. J. Pathol.
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Swigut, T., Alexander, L., Morgan, J., Lifson, J., Mansfield, K. G., Lang, S., Johnson, R. P., Skowronski, J., Desrosiers, R.
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Borda, J. T., Alvarez, X., Kondova, I., Aye, P., Simon, M. A., Desrosiers, R. C., Lackner, A. A.
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Yoshino, N., Lu, F. X.-S., Fujihashi, K., Hagiwara, Y., Kataoka, K., Lu, D., Hirst, L., Honda, M., van Ginkel, F. W., Takeda, Y., Miller, C. J., Kiyono, H., McGhee, J. R.
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Blancou, P., Chenciner, N., Fang, R. H. T., Monceaux, V., Cumont, M.-C., Guetard, D., Hurtrel, B., Wain-Hobson, S.
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Cole, K. S., Steckbeck, J. D., Rowles, J. L., Desrosiers, R. C., Montelaro, R. C.
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Lorin, C., Mollet, L., Delebecque, F., Combredet, C., Hurtrel, B., Charneau, P., Brahic, M., Tangy, F.
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Pistello, M., Matteucci, D., Bonci, F., Isola, P., Mazzetti, P., Zaccaro, L., Merico, A., Del Mauro, D., Flynn, N., Bendinelli, M.
(2003). AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Protection from an Intraclade Challenge Administered Systemically or Mucosally by an Attenuated Vaccine. J. Virol.
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Li, F., Craigo, J. K., Howe, L., Steckbeck, J. D., Cook, S., Issel, C., Montelaro, R. C.
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Alexander, L., Illyinskii, P. O., Lang, S. M., Means, R. E., Lifson, J., Mansfield, K., Desrosiers, R. C.
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Abstract
Introduction
