JOURNAL OF VIROLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Oct. 1999, p. 8356–8363Vol. 73, No. 10
Protection by Live, Attenuated Simian Immunodeficiency Virus
against Heterologous Challenge
MICHAEL S. WYAND,1KELLEDY MANSON,1DAVID C. MONTEFIORI,2JEFFREY D. LIFSON,3
R. PAUL JOHNSON,4AND RONALD C. DESROSIERS4*
New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-91024;
Primedica, Worcester, Massachusetts 016081; Duke University Medical Center, Department of Surgery, Durham, North
Carolina 277102; and AIDS Vaccine Program, SAIC—Frederick, NCI—Frederick Cancer Research
and Development Center, Frederick, Maryland 217013
Received 3 May 1999/Accepted 9 July 1999
We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV),
SIVmac239?3, which is missing nef and vpr genes, to protect against challenge by heterologous strains
SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been
replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are
derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate
lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge
with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria
of CD4?cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of
four vaccinated monkeys despite the complete mismatch of env sequences. However, SHIV89.6p infection was
established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads
between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other
vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads
were lower than those observed in unvaccinated controls but the level of protection was less than what was
observed against SHIV89.6p in these experiments and considerably less than the level of protection against
SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection
by live, attenuated SIVmac239?3 against heterologous virus challenge and suggest that even live, attenuated
vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation
present in field strains of virus. The results further suggest that factors other than anti-Env immune responses
can be principally responsible for the vaccine protection by live, attenuated SIV.
Infection by live, attenuated deletion mutants of simian im-
munodeficiency virus (SIV) has afforded rhesus monkeys
strong protection against subsequent challenge by wild-type,
disease-causing strains of SIV (4, 7, 18, 36). While it has been
argued that this protection could result from some sort of
blocking or interference phenomenon (13, 32, 34), several lines
of circumstantial evidence suggest that the protection may be
immune mediated (16). Protection has been shown to be time
dependent: the longer the time interval, the better the protec-
tion, despite the fact that vaccine virus loads are highest during
the initial weeks following immunization (4, 36). While the
most solid protection has been observed in monkeys with ap-
parent sterilizing immunity, some animals have exhibited long-
term protective effects despite a transient take of the challenge
virus (18, 36), again apparently more consistent with immune-
mediated protection than with viral interference. Monkeys that
control replication of SIVmac239?nef least effectively are the
least protected upon subsequent challenge (23). Finally, CD4?
cells in peripheral blood mononuclear cells (PBMC) of vacci-
nated animals are perfectly capable of supporting the replica-
tion of SIV in culture (11).
Despite these arguments in support of immune-mediated
mechanisms for the protection, no clear evidence has been
forwarded regarding what these immune responses might be.
There is considerable interest in the scientific community in
identifying the responses that are protective in this system
because even if the live, attenuated approach is never for-
warded for trials in humans, such research in monkeys may be
able to tell us what types of immune responses are needed for
protection. In addition, it is not known how broadly protective
the live, attenuated vaccine approach can be against patho-
genic, heterologous virus challenge in this rhesus monkey
model. We have addressed these issues by vaccinating rhesus
monkeys with the attenuated strain SIVmac239?3 (9, 36) and
subsequently challenging them with pathogenic, uncloned, het-
erologous strains SHIV89.6p (28) and SIVsmE660 (12, 15).
MATERIALS AND METHODS
Animals. Rhesus monkeys were received from the Oregon Regional Primate
Research Center, Beaverton, Oreg., or Laboratory Animal Breeders and Ser-
vices, Yemassee, S.C. Upon receipt, the monkeys underwent a 6-week quaran-
tine. During this period, the animals received three intradermal tuberculin tests,
sampling for hematology and serum chemistry profiles, and rectal swabs for
bacterial culture. Feces were also analyzed for occult blood, ovum, and parasite
determinations. In addition, each animal was screened for antibody status with
respect to SIV, simian type D virus, simian T-cell lymphotropic virus type 1,
herpesvirus B, and measles virus. All animals were antibody negative for SIV,
simian type D virus, and simian T-cell lymphotropic virus type 1. All animal care
and use procedures conformed to the revised Public Health Service Policy on
Humane Care and Use of Laboratory Animals.
Vaccination and challenge. The preparation of SIVmac239?3 vaccine stock
has been described previously (9, 36). By intravenous inoculation, four male
rhesus monkeys were vaccinated with SIVmac239?3 containing 0.01 ng of p27
and four were vaccinated with SIVmac239?3 containing 1.1 ng of p27. These
amounts of this stock represent 23 tissue culture infectious doses and 5 rhesus
monkey infectious doses in the first group and 2.3 ? 103tissue culture infectious
* Corresponding author. Mailing address: New England Regional
Primate Research Center, Harvard Medical School, One Pine Hill Dr.,
Box 9102, Southborough, MA 01772-9102. Phone: (508) 624-8042.
Fax: (508) 624-8190. E-mail: firstname.lastname@example.org.
doses and 5 ? 102rhesus monkey infectious doses in the second group. The
preparations of the SIVsmE660 challenge stock (provided by Vanessa Hirsch)
and of the SHIV89.6p challenge stock (provided by Norman Letvin) have been
described (12, 15, 28). Monkeys in the first group were challenged intravenously
with 20 tissue culture infectious doses of SIVsmE660 28 months after the time of
vaccination. Monkeys in the second group were challenged intravenously with
SHIV89.6p containing 0.019 ng of p27 37 months after the time of vaccination.
Flow cytometry. Whole blood collected in EDTA was analyzed for expression
of CD4 (OKT4a [Ortho] and Anti-Leu 3a [Becton Dickinson]), CD8 (Anti-Leu
2a [Becton Dickinson]), and CDw29 (4B4 [Coulter Immunology]) by a whole-
blood lysis technique previously described (36). Briefly, antibody (volume de-
pendent upon the specific antibody) was added to 100 ?l of whole blood and
incubated for 10 min in the dark. Lysing solution (Becton Dickinson) was added
and the samples were incubated for 10 min at room temperature. Stained cells
were fixed with 0.5% paraformaldehyde. Samples were analyzed on a Becton
Dickinson FACScan cytometer.
Cell-associated virus loads. Cell-associated virus loads were determined by
quantitating the numbers of infectious cells in PBMC as previously described (9).
Twelve serial 1:3 dilutions of PBMC, beginning with 106cells, were cocultured in
duplicate with 105CEM?174 cells per well in 24-well plates. Supernatant sam-
ples were collected after 21 days of culture and stored frozen at ??70°C until
analysis for p27 antigen with the Coulter p27 antigen assay kit.
Antiviral antibodies by ELISA. Enzyme-linked immunosorbent assay (ELISA)
plates were coated with purified lysed SIVmac251, SIVmac239, or SIVsmE660 as
previously described (6, 9). Human immunodeficiency virus type 1 (HIV-1) and
SIV envelope proteins were purchased from Immunodiagnostics (Bedford,
Mass.) and also used to coat ELISA plates. Dilutions of sera from the monkeys
were assayed for antibody binding by using an alkaline phosphatase-conjugated
goat anti-human immunoglobulin G (heavy and light chain) in accordance with
our previously described procedures (6, 9).
Plasma RNA loads. Citrated plasma samples were assayed for SIV RNA levels
as described previously (9, 35).
DNA PCR. Genetic analysis of mutant versus wild-type SIV sequences was
performed by using a PCR method previously described (21, 36), with slight
modifications. One-half microgram of chromosomal DNA was used per 100-?l
reaction. DNA was added to the reaction mixture minus Taq polymerase and
denatured at 95°C for 10 min and immediately placed on ice. The DNA-reaction
mix was briefly centrifuged and returned to ice. The DNA-reaction mix was
placed in a thermocycler preheated to 72°C for 1 to 2 min; Taq polymerase (2.5
U) was added and the PCR was carried out for 35 cycles of 94°C for 1 min, 57°C
for 1 min, and 72°C for 1 min 45 s. The analyses for SIVmac239?3 and E660
sequences were conducted separately. The primer pairs for SIVmac239?3 were
as previously described (36). The sequences of the forward primers for E660
were PCR1 (5?-ACA ACA AAA CAT GGA TGA TGT GG) and PCR2 (5?-
AGT CCC CTT AAG GGC CAT GAC ATA).
HIV-1 env sequences were amplified similarly using HIV-1 89.6 env-specific
primer pairs. The expected size for the first round of PCR was 962 bp (primers
were F1 [GCAACCACCACTCTATTTTGTGC] and R1 [CCTCCTGAGGAT
TGATTAAAGGC]). The expected size for the nested PCR was 453 bp (primers
were F2 [GGATGAAAGCCTAAAGCCATGTG] and R2 [AGCAGTTGAGT
TGACACCACTGG]). Ten microliters was electrophoresed in a 1.5% agarose
gel containing ethidium bromide.
Virus neutralization assays. Neutralizing antibodies were measured in either
CEM?174 cells (SIVmac251LP and SIVsmE660) or MT-2 cells (HIV-1 MN and
SHIV-89.6p) by a reduction in virus-induced cell-killing effects as described
previously (4, 15). Assay stocks of SIVmac251LP, SIVsmE660, and HIV-1 MN
were prepared in H9 cells and SHIV-89.6p was prepared in human PBMC. Titers
are reported as the reciprocal serum dilutions at which 50% of cells were
protected from virus-induced killing. This cutoff usually corresponds to a ?90%
reduction in viral Gag antigen synthesis.
Assay of CTL activity. SIV-specific cytotoxic T lymphocytes (CTL) were ana-
lyzed following antigen-specific stimulation of PBMC as described previously
(17). Briefly, PBMC were stimulated by using autologous herpesvirus papio-
transformed 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, which were inactivated with UV psoralen after overnight incubation. After
10 to 12 days of culture, stimulated PBMC were used as effector cells in a
standard51Cr-release assay. Target cells consisted of autologous B-LCL infected
with recombinant vaccinia viruses expressing SIV proteins. Recombinant vac-
cinia viruses used to infect target cells include vAbt252 (encoding the SIVmac251
p55 Gag and protease proteins; Therion), rVV-239 (encoding the SIVmac239
envelope [Env]) (30), and the control vaccinia virus NYCBH. Cold targets
consisting of unlabeled autologous B-LCL infected with the control vaccinia
virus NYCBH were used at a cold/hot target ratio of 15:1 to decrease background
lysis. Chromium released was assayed after a 5-h incubation at 37°C in a 5% CO2
incubator, and the percent cytotoxicity was calculated as follows: (test release ?
spontaneous release)/(maximum release ? spontaneous release) ? 100. SIV-
specific release was then calculated by subtracting lysis of control NYCBH-
infected target cells from that of target cells expressing SIV antigens. Based on
examination of 10 naive control animals not infected with SIV, SIV-specific lysis
of greater than 5% seen at more than one effector/target ratio was interpreted as
Vaccine phase. Eight monkeys were vaccinated by intrave-
nous inoculation with SIVmac239?3, which contains two de-
letions in nef and a deletion in vpr (9). The response of rhesus
monkeys to the SIVmac239?3 was similar to what has been
described previously (9). There was an early spike in virus load
around 2 to 3 weeks after inoculation, which subsequently
resolved (Fig. 1A and B). Anti-SIV antibody responses were
readily detected (Fig. 1C and D) and these persisted for the
duration of the vaccine phase. There were no statistically sig-
nificant differences in either peak viral PBMC loads or levels of
SIV antibodies at 44 weeks between the groups of monkeys
vaccinated with SIVmac239?3 containing 0.01 versus 1.1 ng of
p27 (P ? 0.3; Mann-Whitney test).
SHIV89.6p challenge. SHIV89.6 is a recombinant virus
which contains gag and pol sequences from SIVmac239 and env
sequences from HIV-1 isolate 89.6 (29). SHIV89.6p is a highly
aggressive derivative that causes acute declines in CD4?cell
numbers and death with AIDS over a time course that is
usually less than 12 weeks (28). Thus, challenge in this case was
with an uncloned strain of virus very closely matched in gag and
pol sequences (19) but totally mismatched in env sequences
(Table 1). Challenge was performed by the intravenous route
with SHIV89.6p containing 0.019 ng of p27gagantigen 37
months after the time of vaccination. Two naive rhesus mon-
keys served as controls for challenge.
Both unvaccinated monkeys inoculated with SHIV89.6p de-
veloped rapid, severe declines in CD4 cell numbers (Fig. 2A).
CD4?cell counts were reduced to near zero by 5 weeks (Fig.
2A). These control animals were euthanized at 8 weeks post-
inoculation because of deteriorating clinical condition. At nec-
ropsy, one animal had Epstein-Barr virus esophagitis and lym-
phoid hyperplasia and the other animal had lymphoid follicular
involution and inflammatory infiltrates in the gastrointestinal
tract. This acute clinical course is typical of pathogenic
SHIV89.6p infections that we have observed in four other
monkeys inoculated with the same stock of virus. In contrast to
these control animals, all four of the vaccinated monkeys were
completely protected against any CD4 cell depletion (Fig. 2A).
Vaccine virus was not recovered from PBMC of any of the
four vaccinated monkeys at the time of challenge (Fig. 2B) or
during the weeks preceding challenge, even when 106PBMC
were used for cocultivation. These findings are consistent
with previous descriptions of the attenuated nature of SIV-
mac239?3 (9). Cell-associated virus loads, measured as the
numbers of infectious cells in PBMC, peaked in the control
animals around 2 weeks after challenge and, following a slight
decline, remained at moderate or high levels until the time of
death (Fig. 2B). Virus was recovered from only one of the four
vaccinated animals at week 2 postinoculation. In fact, cell-
associated viral loads remained low in all four vaccinated
animals throughout the remaining course of measurements
Viral loads were also evaluated after challenge by quantitat-
ing the amounts of virion-associated viral RNA in plasma.
Viral RNA was detected in plasma for three of the four vac-
cinated monkeys at week 2 following challenge (Fig. 2C). How-
ever, the levels of viral RNA at week 2 postchallenge were 2 to
4 logs lower in these three vaccinated animals than in the two
controls (Fig. 2C). The levels of viral RNA declined to unde-
tectable levels by week 8, but they persisted at fluctuating levels
of 1,000 to 10,000 RNA copy equivalents per ml of plasma in
VOL. 73, 1999 PROTECTION BY LIVE, ATTENUATED SIV 8357
three of the four vaccinated animals between 30 and 72 weeks
postchallenge (Fig. 2C). The one animal (17170) that had no
detectable viral RNA in plasma in the initial weeks following
challenge is the same animal that maintained undetectable
levels of plasma RNA 30 to 72 weeks after challenge (Fig. 2C).
DNA was prepared from PBMC obtained at day 15 and
weeks 20, 32, and 70 from the SHIV-challenged monkeys.
DNA was used for genetic analysis of the presence of HIV-1
89.6 env sequences by PCR. As expected, day 15 PBMC from
both unvaccinated control animals yielded a fragment consis-
tent with the presence of SHIV89.6 challenge virus (Table 2).
The two vaccinated monkeys with significant viral loads at 2
weeks after the SHIV challenge (17157 and 17049) also
showed HIV-1 env sequences with the day 15 sample (Table 2).
The other two vaccinated monkeys did not have detectable
HIV-1 env sequences in the day 15 PBMC sample (Table 2).
None of the four vaccinated monkeys had detectable HIV-1
env sequences in the week 20 PBMC sample. However, all four
of the previously vaccinated monkeys had HIV-1 env se-
quences detectable in their PBMC at both 32 and 70 weeks
after the SHIV89.6p challenge (Table 2).
SIVsmE660 challenge. SIVsmE660 (12) is an independent
isolate of SIVsm, unlinked by any recent history to SIVmac239
or SIVmac251. Infection of rhesus monkeys with SIVsmE660
results in consistently high viral loads and eventual progression
to AIDS and death (15). Thus, challenge in this case was with
an uncloned strain of SIV within the same grouping of primate
lentiviruses as SIVmac but with natural sequence variation
throughout its genome. Although SIVsmE660 has not been
directly sequenced, it is closely related to SIVsmH4 and
SIVsmE543-3 since all were derived from the same original
infected animal (14, 15). SIVsmH4 and SIVsmE543-3 have
been completely sequenced; they are very similar, and the
relatedness of their sequences to SIVmac239 has been previ-
ously calculated (14). We can thus estimate 89 to 92% amino
acid identity in gag-pol and about 82% identity in env between
FIG. 1. Vaccine phase. (A) Cell-associated vaccine virus loads following vaccination in monkeys that were subsequently challenged with SHIV89.6p. (B) Cell-
associated vaccine virus loads following vaccination in monkeys that were subsequently challenged with SIVsmE660. Code for PBMC load: 0, virus was not recovered
with 106or fewer PBMC; 1, virus was recovered with 106but 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. (C) Anti-SIV antibody responses as detected by ELISA following vaccination in monkeys that were subsequently challenged with
SHIV89.6p. (D) Anti-SIV antibody responses as detected by ELISA following vaccination in monkeys that were subsequently challenged with SIVsmE660. A410,
absorbance at 40 nm.
TABLE 1. Sequence divergence and protection
% Amino acid identity
% Amino acid identity
aCompared to vaccine strain SIVmac239?3.
b37 months after the time of vaccination. ???, strong but incomplete.
c28 months after the time of vaccination. ?, weak.
8358 WYAND ET AL.J. VIROL.
SIVsmE660 and SIVmac239 (Table 1). Challenge was per-
formed by the intravenous route with 20 tissue culture infec-
tious doses of virus 28 months from the time of SIVmac239?3
vaccination. Two naive rhesus monkeys again served as con-
trols for the challenge.
Vaccine virus was not recovered from three of the four
vaccinated monkeys around the time of challenge, and it was
recovered from the fourth only when 106PBMC were used
(Fig. 3A). Cell-associated virus loads in the control animals
reached high levels in the weeks immediately following chal-
lenge and remained at high levels until the time of their deaths
with AIDS 50 to 60 weeks following inoculation (Fig. 3A). SIV
was recovered from three of the four vaccinated animals on
multiple occasions over the first 10 weeks with 333,333 or fewer
PBMC (Fig. 3A). Cell-associated loads subsequently declined
to low levels, with no virus recovered even with 106PBMC in
three of the four vaccinated monkeys between 18 and 65 weeks
postchallenge. The fourth vaccinated monkey (17155), how-
ever, maintained high cell-associated viral loads for the dura-
tion of the measurements.
The results of plasma RNA measurements in this study also
agreed well with the cell-associated viral load measurements.
Plasma RNA in 17155 increased to levels approximating those
in the control animals (Fig. 3B). Two of the three remaining
vaccinated animals (17052 and 17158) maintained persistently
detectable plasma RNA levels for at least 24 weeks, but the
levels were approximately 2 to 3 logs lower than those seen in
the unvaccinated controls (Fig. 3B).
Both of the unvaccinated controls exhibited declines in CD4
cell numbers up until the time of their deaths (Fig. 3C). At
necropsy, neither of the two control animals had opportunistic
infections. However, animal KJ8 had diffuse follicular hyper-
plasia with multiple lymphoid nodules characteristic of lym-
phoproliferative disease, and animal VT2 had lymphoprolif-
erative disease and SIV arteriopathy. Only one of the four
vaccinated animals (17052) has remained alive with normal
CD4 cell numbers (Fig. 3C). Three of the four vaccinated
animals were euthanized due to clinical deterioration 50 to 60
weeks following inoculation. All three of these animals had
decreased CD4 cell percentages (Fig. 3C). At necropsy, animal
17155 had marked diffuse follicular hyperplasia and dissemi-
nated lymphoid nodules characteristic of lymphoproliferative
disease. Animal 17158 had an opportunistic infection with
cryptosporidiosis in the small and large intestine as well as
lymphocytic meningoencephalitis. The third vaccinated animal
that died, 17159, had glomerulonephritis, thymic dysinvolution,
SIV arteriopathy, and rare multinucleate giant cells in the
pleura of the lung. The decreases in CD4 numbers, clinical
condition, and necropsy findings are compatible with SIV-
Nested PCR was used to examine viral DNA sequences
present in PBMC at 15 days, 22 weeks, and 49 weeks after
challenge. Primers spanning the nef gene specific for
FIG. 2. Outcome of challenge with SHIV89.6p. (A) CD4?T lymphocytes.
(B) Cell-associated virus loads measured as the number of infectious cells in
PBMC. Code is as used previously (9, 36): 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. (C) Viral RNA levels in plasma. Open symbols used for control animals
EPF and EPJ. Closed symbols are for vaccinated animals. The detection limit,
approximately 300 copy equivalents (Copy Eq) per ml, is indicated by the dashed
TABLE 2. HIV-1 env sequences in PBMC after
Day 15Week 20 Week 32Week 70
aNA, sample not available because of death of the animal. EPF and EPJ are
the control unvaccinated monkeys. Each sample was analyzed in triplicate and
triplicate samples were either all positive (?) or all negative (?) as indicated.
Primers specific for a conserved region of HIV 89.6 env were used as described
in Materials and Methods.
VOL. 73, 1999PROTECTION BY LIVE, ATTENUATED SIV8359
FIG. 3. Outcome of challenge with SIVsmE660. (A) Cell-associated virus loads measured by the number of infectious cells in PBMC. Code for PMBC load: 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. (B) Viral RNA loads in plasma. (C) CD4?T
lymphocytes. Open symbols are for control unvaccinated animals VT2 and KJ8. Closed symbols are for vaccinated animals.
8360WYAND ET AL.J. VIROL.
SIVmac239 and SIVsmE660 sequences were used. For the
control unvaccinated animals, fragments corresponding to full-
length challenge virus were all that were detected (Table 3).
Using PBMC from the four vaccinated monkeys, SIV239-spe-
cific primers detected either no viral sequences or viral se-
quences corresponding to the length of the vaccine strain in 11
of the 12 samples examined (Table 2). The SIVsmE660-spe-
cific primers detected challenge virus in all four vaccinated and
challenged animals at one or more of the three time points
examined (Table 3). Vaccinated monkey 17159, which main-
tained the lowest viral loads after challenge (Fig. 3B), was
negative for the detection of viral sequences with both sets of
primers at weeks 22 and 49 (Table 3).
Immune responses. Serum taken just prior to the time of
challenge was used for measurement of SIV-specific antibody
responses. Ability to neutralize a laboratory-passaged stock of
SIVmac251, an uncloned virus closely related in sequence to
SIVmac239 (3), around the time of challenge was similar in the
two groups: a median of 1:459 in the SHIV-challenged group
and 1:685 in the SIVsmE660-challenged group (Table 4).
Three of the four animals challenged initially with SIVsmE660
showed weak neutralizing activity against SIVsmE660 at the
time of challenge (Table 4). The four animals challenged with
SHIV89.6p showed no detectable neutralizing activity against
SHIV89.6p or against HIV-1MN (Table 4). The lack of neu-
tralizing activity against HIV-1 and SHIV is not surprising
given the very low level of amino acid similarity in env com-
pared to SIVmac239?3 (Table 1).
CTL activity against SIVgagand SIVenvantigens was also
assessed by using blood samples taken on the day of challenge.
Three of the four SHIV-challenged animals showed significant
CTL activity to SIVmac251 antigens on the day of challenge as
assessed by percent specific lysis (Fig. 4). 17157, the animal
without measurable anti-Gag CTL activity, is the one animal
with a spike in recoverable virus at week 2 after challenge (Fig.
2B) and this animal also had HIV-1 env sequences detectable
in PBMC at week 2 after challenge (Table 2). CTL activity
against SIVmac251 antigens in the SIVsmE660-challenged an-
imals assessed on the day of challenge was as high as or higher
than in the SHIV-challenged animals (Fig. 5). The animal with
the lowest level of SIV-specific CTL activity prechallenge
(17158) had the highest peak level of viremia, while the animal
with the highest CTL activity (17155) had the lowest peak
viremia. CTL activity against SIVsmE660 antigens was not
assessed prior to challenge.
Our results demonstrate significant but incomplete protec-
tion afforded by SIVmac239?3
SHIV89.6p. All four vaccinated animals were strongly pro-
tected against the rapid CD4 declines and the development of
disease. One of the four vaccinated and challenged monkeys
exhibited viral load set points below the limits of detection and
the other three exhibited fluctuating levels between cutoff
(?300 copy equivalents of viral RNA per ml of plasma) and
10,000 copy equivalents of viral RNA per ml of plasma. None
of the vaccinated animals exhibited apparent sterilizing immu-
nity against the SHIV89.6p challenge. While there have been
FIG. 4. CTL activity to SIVmac antigens (Gag and Env) on the day of
challenge with SHIV89.6p. Assays were performed following antigen-specific
stimulation of PBMC (17) at the indicated effector/target ratios.
TABLE 4. Virus-neutralizing activity in sera from SIVmac239?3-
vaccinated monkeys at the time of challengea
Result following vaccination with:
aSera taken within 24 h prior to challenge were used in virus neutralization
assays. Activities are presented as the reciprocal of serum dilutions at which 50%
of the cells were protected.
bSIVmac251LP, a laboratory-passaged stock of SIVmac251.
cNT, not tested.
TABLE 3. Viral sequences in PBMC after SIVsmE660 challenge
Day 15Week 22Week 49
aPCR primers corresponding to sequences specific for SIVmac239 and
SIVsmE660 that span nef were used (see Materials and Methods).
bNeg, viral sequences were not detected. ND, not done. WT, PCR product
corresponding to full-length, nondeleted, wild-type challenge virus was detected.
VOL. 73, 1999 PROTECTION BY LIVE, ATTENUATED SIV8361
previous reports of protection against SHIV by live, attenuated
SIV, most have used nonpathogenic SHIV (1, 5, 33). Our
results, and those in a very recent publication (23), extend the
descriptions of protective effects of live, attenuated SIV to
more highly stringent challenge with an aggressive, pathogenic
SHIV. Our findings are consistent not only with the recent
report of Lewis et al. (23) but also with reports on the ability of
nonpathogenic SHIVs to protect against challenge by patho-
genic SIVmac or SIVsm (24, 27).
Vaccination with SIVmac239?3 was also only partially pro-
tective against SIVsmE660 challenge when performed in a
similar format. Protection against SIVsmE660 was actually less
impressive than protection against SHIV89.6p in the limited
number of animals studied. No significant protection was ob-
served in one of the vaccinated, E660-challenged animals and
the other three exhibited 2- to 4-log reductions in viral load at
set point compared to control unvaccinated animals. Nonethe-
less, only one of these four vaccinated, E660-challenged mon-
keys remains alive with normal CD4 counts. The level of pro-
tection afforded by SIVmac239?3 against E660 challenge
appears to be much lower than what has been observed pre-
viously for similar intravenous challenge with SIVmac251 (36).
SIVmac251 exhibits a pattern of disease and time course in
naive rhesus monkeys similar to that of SIVsmE660, but SIV-
mac251 is much more closely related in sequence to the vac-
cine strain, SIVmac239?3, than is SIVsmE660. SIVmac251 is
related in passage history to SIVmac239 (8, 20) and is only
slightly heterologous (3). Thus, as difficult as it will be to match
the live, attenuated approach for protective efficacy, even the
live, attenuated approach will face significant hurdles in pro-
viding protection against the natural variation present in field
strains of virus.
Because gp120/gp41 Env proteins are the only virus-encoded
proteins on the surface of viral particles and since neutralizing
antibodies are directed to them, it has generally been assumed
that anti-Env immune responses would be important for pro-
viding protection. Our results indicate that factors other than
anti-Env immune responses can be principally responsible for
the vaccine protection by SIVmac239?3, at least against SHIV
challenge. The sequences of the env genes of HIV-1 and those
of SIVmac/SIVsm are highly divergent. Inspection of their
predicted amino acid sequences reveals identical stretches no
greater than six amino acids in length. We found that antibod-
ies raised to the SIV vaccine strain did not react appreciably to
HIV-1 env (data not shown) and did not neutralize HIV-1
infectivity detectably (Table 4), similar to previous reports (25,
31, 33). In contrast, antibodies raised to the SIV vaccine strain
did react well with SIVsmE660 (data not shown) and they were
able to neutralize SIVsmE660 infectivity at least to some ex-
tent (Table 4). Nonetheless, protection against SIVsmE660
was no better than, and perhaps even worse than, the protec-
tion against SHIV89.6p. In contrast to the minimal degree of
matching of env sequences, the gag-pol sequences of SHIV89.6
are ?99.5% identical with gag-pol sequences of SIVmac239?3
(Table 1) (19).
The results described in this report and in another recent
manuscript from our group (18) focus attention on the po-
tential of cellular responses, particularly CTL, to Gag and
Pol for controlling SIV and SHIV infection. In the recent
study (18), monkeys were vaccinated with SIVmac239?3X and
SIVmac239?4 and were challenged vaginally. The develop-
ment of early or stronger SIV-specific CTL responses ap-
peared to be associated with protection and, in the ?4-immu-
nized monkeys, protection was observed in the absence of
detectable neutralizing antibodies and in one case with only
extremely low levels of binding antibodies detectable only with
sensitive tests (18). The importance of CTL responses is con-
sistent with the extreme difficulty in neutralizing primary iso-
lates of SIV and HIV (2), with evidence suggesting a role for
CTL in controlling viremia during natural infection (22, 26),
and with the ability of vaccine-induced anti-Nef CTL to sup-
press viral replication in some cases following challenge (10).
This of course does not exclude the possibility that antibodies
could provide complete protection on their own in some cir-
cumstances or that antibodies could contribute to the protec-
tive capacity of live, attenuated vaccine approaches.
We thank Marı ´a Garcı ´a-Moll of Bio-Molecular Technology, Inc., for
the genetic analyses; Michael Piatak, Jr., Li Li, and Tom Parks for
plasma RNA analyses; and Rhona Glickman for the CTL assays. We
thank Vanessa Hirsch and Norman Letvin for providing the challenge
virus stocks. We also thank Susan Czajak for technical assistance and
help with figures and Joanne Newton and Jane FitzPatrick for manu-
This work has been supported by PHS grants AI35365 and RR
00168, NIAID contract N01-AI-65303, and federal funds from the
National Cancer Institute, National Institutes of Health, under con-
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