JOURNAL OF VIROLOGY, Apr. 2005, p. 4580–4588
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 8
Vaccine-Elicited Memory Cytotoxic T Lymphocytes Contribute to
Mamu-A*01-Associated Control of Simian/Human
Immunodeficiency Virus 89.6P Replication
in Rhesus Monkeys
Michael S. Seaman,1Sampa Santra,1Michael H. Newberg,1Valerie Philippon,2
Kelledy Manson,2Ling Xu,3Rebecca S. Gelman,4Dennis Panicali,2
John R. Mascola,3Gary J. Nabel,3and Norman L. Letvin1,3*
Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School,1and Department of
Biostatistics, Dana-Farber Cancer Institute,4Boston, and Therion Biologics, Cambridge,2Massachusetts, and
Vaccine Research Center, National Institutes of Allergy and Infectious Disease,
National Institutes of Health, Bethesda, Maryland3
Received 18 October 2004/Accepted 6 December 2004
The expression of particular major histocompatibility complex (MHC) class I alleles can influence the rate
of disease progression following lentiviral infections. This effect is a presumed consequence of potent cytotoxic
T-lymphocyte (CTL) responses that are restricted by these MHC class I molecules. The present studies have
examined the impact of the MHC class I allele Mamu-A*01 on simian/human immunodeficiency virus 89.6P
(SHIV-89.6P) infection in unvaccinated and vaccinated rhesus monkeys by exploring the contribution of
dominant-epitope specific CTL in this setting. Expression of Mamu-A*01 in immunologically naive monkeys
was not associated with improved control of viral replication, CD4?T-lymphocyte loss, or survival. In contrast,
Mamu-A*01?monkeys that had received heterologous prime/boost immunizations prior to challenge main-
tained higher CD4?T-lymphocyte levels and better control of SHIV-89.6P replication than Mamu-A*01?
monkeys. This protection was associated with the evolution of high-frequency anamnestic CTL responses
specific for a dominant Mamu-A*01-restricted Gag epitope following infection. These data indicate that
specific MHC class I alleles can confer protection in the setting of a pathogenic SHIV infection by their ability
to elicit memory CTL following vaccination.
The expression of certain major histocompatibility complex
(MHC) class I alleles influences disease progression following
human immunodeficiency virus type 1 (HIV-1) and simian
immunodeficiency virus (SIV) infection. Expression of HLA-
B*35 and HLA-Cw*04 has been linked to rapid disease pro-
gression (4), whereas expression of HLA-B*27 and HLA-B*57
is associated with slow disease progression in HIV-1-infected
humans (8, 14). Similarly, expression of the MHC class I alleles
Mamu-A*01 and Mamu-B*17 in Indian origin rhesus monkeys
has been associated with slow disease progression following
infection with the pathogenic virus SIVmac251, SIVsmE660,
or SIVmac239 (15, 16, 20, 21). It is presumed that the im-
proved survival of these virus-infected humans and monkeys
reflects the contribution of MHC class I-restricted epitope-
specific cytotoxic T-lymphocyte (CTL) responses. Immuno-
dominant Gag-specific CTL responses restricted by HLA-B*27
and HLA-B*57 have been demonstrated for individuals chron-
ically infected with HIV-1 who have slowly progressive disease
(6, 7, 18). Further, Mamu-A*01- and Mamu-B*17-restricted
dominant-epitope-specific CTL responses in rhesus monkeys
have been associated with slow disease progression following
SIVmac239 infection (20).
Virus-specific CTLs also play a central role in containing
viral replication in vaccinated monkeys that are subsequently
challenged with primate immunodeficiency viruses. Vaccine-
elicited memory CTLs have been shown to expand in monkeys
following viral infection and to contribute to protection against
the progression of clinical disease (3, 10, 21, 23, 27). This
vaccine-generated immunity controls the extent of viral repli-
cation and diminishes CD4?T-lymphocyte loss in infected
animals. Whether MHC class I haplotype can influence this
vaccine-mediated protection has not been systematically ex-
Chimeric simian/human immunodeficiency viruses (SHIVs)
are widely used as challenge viruses in nonhuman primate
studies of HIV-1 vaccine strategies (1–3, 5, 10, 13, 23, 27).
Infection of rhesus monkeys with the CXCR4-tropic, highly
(SHIV-89.6P) virus is associated with acute loss of CD4?T
lymphocytes, high levels of persistent viral replication, and
rapid progression to AIDS and death (22). Conflicting data
have been reported concerning the impact of particular MHC
class I alleles on disease progression in SHIV-89.6P-infected
rhesus monkeys. Some investigators have reported that Mamu-
A*01 expression is associated with a more benign clinical
course (29), while others have observed no impact on disease
evolution in previously naive animals (21).
In the present study, we analyzed virus replication, CD4?
T-lymphocyte loss, and Gag-specific cellular immune re-
sponses in unvaccinated and vaccinated Mamu-A*01?and
* Corresponding author. Mailing address: Beth Israel Deaconess
Medical Center, Division of Viral Pathogenesis, 330 Brookline Ave.
RE-113, Boston, MA 02215. Phone: (617) 667-2766. Fax: (617) 667-
8210. E-mail: firstname.lastname@example.org.
Mamu-A*01?rhesus monkeys following SHIV-89.6P infec-
tion. Expression of Mamu-A*01 did not confer a clinical ben-
efit on SHIV-89.6P-induced disease in previously naive mon-
keys. In contrast, expression of this MHC class I allele was
associated with improved control of SHIV-89.6P infection in
monkeys immunized prior to infection. This viral control cor-
related with the development of a high-frequency dominant-
epitope Gag-specific CTL response following viral challenge.
MATERIALS AND METHODS
Selection of rhesus monkeys. A PCR-based assay was used to select adult
rhesus monkeys (Macaca mulatta) that expressed the Mamu-A*01 MHC class I
allele. Briefly, DNA was extracted from peripheral blood mononuclear cells
(PBMC) by using a QIAmp blood kit (QIAGEN, Chatsworth, Calif.). PCR
amplification was then performed using Mamu-A*01-specific primers (forward,
5? GAC AGC GAC GCC GCG AGC CAA 3?; reverse, 5? CGC TGC AGC GTC
TCC TTC CCC 3?). Two additional primers specific for a conserved MHC class
II sequence were also included as internal positive controls (forward, 5? GCC
TCG AGT GTC CCC CCA GCA CGT TTC 3?; reverse, 5? GCA AGC TTT
CAC CTC GCC GCT G 3?). Electrophoresis on a 2% agarose gel yielded a
685-bp band in Mamu-A*01-positive samples and a 260-bp control band in all
samples. Verification of positive samples was achieved by complete DNA se-
quence analysis and comparison with the published Mamu-A*01 sequence. Mon-
keys expressing the Mamu-A*02 MHC class I allele were identified using Mamu-
A*02 specific primers (forward, 5? GTG GGT GGA GCA GGA GGG TCC A
3?; reverse, 5? CAG CAC CTC AGG GTG GCC TCT 3?) as described above.
Monkeys were housed in facilities accredited by the Association for the Assess-
ment and Accreditation of Laboratory Animal Care in accordance with the
guidelines of the Institutional Animal Care and Use Committee for Harvard
Medical School and the Guide for the Care and Use of Laboratory Animals.
Immunization and challenge of rhesus monkeys. Two cohorts each of unvac-
cinated and vaccinated monkeys were analyzed in this study. The first evaluated
group was comprised of 12 Mamu-A*01?and 16 Mamu-A*01?rhesus monkeys
that received DNA prime/recombinant poxvirus boost immunizations and 9
Mamu-A*01?and 19 Mamu-A*01?sham-immunized control animals (25). Plas-
mid DNA vaccines were administered by intramuscular injection at 0, 4, and 8
weeks by the use of a needle-free Biojector system and a no. 3 syringe. For each
DNA immunization, monkeys received 5 mg of SIVmac239 gag plasmid and 5 mg
of HIV-1 89.6P env plasmid (10 mg total). These monkeys also received 5 mg of
IL-2/Ig plasmid on day 2 after DNA vaccination. On week 42 of the study, the
treated monkeys were divided into four groups (each having three Mamu-A*01?
and four Mamu-A*01?animals) and received boost immunizations with plasmid
DNA or one of the three recombinant poxvirus vectors expressing immunogens
matching those used for the DNA prime. A total of 2 ? 109PFU of recombinant
modified vaccinia virus Ankara, recombinant fowlpox virus, or recombinant
vaccinia virus were delivered by both intradermal and intramuscular injections
(109PFU expressing SIVmac239 Gag and 109PFU expressing HIV-1 89.6P
Env). Control monkeys (9 Mamu-A*01?and 19 Mamu-A*01?) were immunized
with 10 mg of sham DNA at weeks 0, 4, and 8 and boosted with sham DNA or
2 ? 109PFU of empty fowlpox virus, modified vaccinia virus Ankara, or vaccinia
virus vectors at week 42. At week 60, all monkeys received an intravenous
challenge with 50 50% monkey infective doses (MID50) of SHIV-89.6P.
The second group was comprised of 8 Mamu-A*01?and 16 Mamu-A*01?(of
which 7 were Mamu-A*02?) rhesus monkeys that received DNA prime/recom-
binant replication-defective adenovirus (rAd) boost immunizations and 2 Mamu-
A*01?and 4 Mamu-A*01?sham-immunized control animals (26). Plasmid DNA
vaccines were administered by intramuscular injection using a needle-free
Biojector system and a no. 3 syringe (Bioject, Portland, Oreg.) at 0, 4, and 8
weeks. For each DNA immunization, monkeys received 4.5 mg of SIVmac239
gag/pol/nef plasmid and 4.5 mg of HIV-1 env plasmid(s) (9 mg total). For the
HIV-1 Env component of the vaccine, four groups of monkeys (each having two
Mamu-A*01?and four Mamu-A*01?animals) received either 4.5 mg of HXBc2/
BaL clade B env, 1.5 mg of HXBc2/BaL clade B env plus 3.0 mg of sham plasmid,
4.5 mg of clade C env, or a mixture of 1.5 mg each of clade A, B, and C env
plasmids. Plasmid DNAs were divided into two aliquots of 0.5 ml each and
delivered into each quadriceps muscle. At week 26, monkeys received a boost
immunization with rAd vectors expressing immunogens matching those used for
the DNA prime. A total of 2 ? 1012rAd particles (1 ? 1012rAd-SIVmac239
gag/pol and 1 ? 1012rAd-HIV-1 env[s]) were delivered by intramuscular injec-
tion as described above. Control monkeys (two Mamu-A*01?and four Mamu-
A*01?) were immunized with sham DNA at weeks 0, 4, and 8 and boosted with
sham rAd vectors at week 26. At week 42, all monkeys received an intravenous
challenge with 50 MID50of SHIV-89.6P. The same stock of SHIV-89.6P chal-
lenge virus was used for both studies. To investigate the effect of Mamu-A*01
expression on immunologically naive rhesus monkeys infected with SHIV-89.6P,
unvaccinated Mamu-A*01?and Mamu-A*01?monkeys from both studies were
grouped together for analysis. To investigate the effect of Mamu-A*01 expression
on vaccinated monkeys infected with SHIV-89.6P, the groups of Mamu-A*01?
and Mamu-A*01?monkeys receiving either DNA prime/rAd boost or DNA
prime/poxvirus boost immunizations were analyzed separately.
Quantitation of plasma viral RNA levels and peripheral blood CD4?T lym-
phocytes. Plasma viral RNA levels were measured by an ultrasensitive branched
DNA amplification assay (Bayer Diagnostics, Berkeley, Calif.) with a lower
detection limit of 125 copies per ml. The percentage of CD4?T lymphocytes in
the peripheral blood of infected monkeys was determined by monoclonal anti-
body staining and flow cytometric analysis. Briefly, freshly isolated peripheral
blood lymphocytes (PBL) were stained with anti-CD3 allophycocyanin (FN18),
anti-CD4 phycoerythrin (19Thy5D7), and anti-CD8 fluorescein isothiocyanate
(SK1; BD Biosciences, Mountain View, Calif.). Samples were acquired using a
FACSCalibur flow cytometer, and data were analyzed using CellQuest software
(BD Biosciences). For the control animals, RNA levels and CD4?T-lymphocyte
percentages were compared for each available time point up to day 168. The
average set point viral RNA levels and CD4?percentages between days 42 and
168 were also compared. The reason for choosing day 168 as the last observation
boundary of these comparisons and averages was that the majority of unvacci-
nated control animals in both groups remained alive through this time point. The
peak viral loads (which occurred between days 14 and 16 following challenge)
were also compared for the two groups of monkeys. These same comparisons
were done for the vaccinated Mamu-A*01?and Mamu-A*01?monkeys, with the
exception that measurements were compared at all time points up to day 300
following challenge and that the averages were calculated for the period of day
42 to day 300. The reason for choosing day 300 for the vaccinated groups of
monkeys was that all animals were monitored for at least this long for both viral
load and CD4?percentage.
IFN-? ELISPOT assays. Multiscreen 96-well plates (Millipore, Bedford,
Mass.) were coated overnight (100 ?l/well) at 4°C with mouse anti-human
gamma interferon (IFN-?) monoclonal antibody (B27; BD PharMingen, San
Diego, Calif.) at 10 ?g/ml in endotoxin-free Dulbecco’s phosphate-buffered
saline (PBS) (D-PBS; Life Technologies, Gaithersburg, Md.). Plates were
washed three times with D-PBS containing 0.25% Tween 20, blocked for 2 h at
37°C with 100 ?l of D-PBS containing 5% fetal bovine serum/well, and rinsed
with RPMI medium containing 10% fetal bovine serum to remove the Tween 20.
PBL were plated in triplicate at 2 ? 105/well in a 100-?l final volume with
medium alone, Gag peptide pool, 89.6P Env peptide pool, or individual Gag-
derived epitope peptides. The peptide pools covered the entire SIVmac239 Gag
or HIV-1 89.6P Env proteins and were comprised of 15 amino acid peptides
overlapping by 11 amino acids or of 20 amino acid peptides overlapping by 10
amino acids, respectively. Each peptide in the pool was present at a concentra-
tion of 1 ?g/ml. The Gag-derived epitope peptides p11C (CTPYDINQM) and
p17G (GSENLKSLY) were used at 1 ?g/ml for measuring antigen-specific CTL
responses in Mamu-A*01?and Mamu-A*02?rhesus monkeys, respectively. Fol-
lowing an 18-h incubation at 37°C, the plates were washed nine times with D-PBS
containing 0.25% Tween 20 and once with distilled water. The plates were then
incubated with 2 ?g of biotinylated rabbit anti-human IFN-? antibody (Bio-
source, Camarillo, Calif.)/ml for 2 h at room temperature, washed six times with
Coulter Wash (Beckman Coulter, Miami, Fla.), and incubated for 2 h with a
1:500 dilution of streptavidin-AP (Southern Biotechnology, Birmingham, Ala.).
Following five washes with Coulter Wash and one with D-PBS, the plates were
developed with nitroblue tetrazolium–5-bromo-4-chloro-3 indolyl phosphate
chromogen (Pierce, Rockford, Ill.), the enzymatic reaction was stopped by wash-
ing with tap water, and the plates were air dried and read using an enzyme-linked
immunospot (ELISPOT) reader (Hitech Instruments, Edgement, Penn.). The
mean number of spots from triplicate wells was calculated for each animal and
adjusted to represent the mean number of spots per 106PBMC. Negative-control
wells with no peptide antigen consistently had less than 50 spots per 106PBMC.
Tetramer staining analysis. Tetrameric complexes of Mamu-A*01/p11C and
Mamu-A*02/p17G were prepared as previously described (9, 17). PBL were
isolated from whole-blood specimens by Ficoll-Paque (Amersham-Pharmacia
Biotech, Uppsala, Sweden) density gradient centrifugation. Phycoerythrin-cou-
pled tetrameric complexes were used in combination with anti-CD3-allophyco-
cyanin and anti-CD8-fluorescein isothiocyanate to stain 2 ? 105freshly isolated
PBL. Samples were acquired using a FACSCalibur flow cytometer, and data
were analyzed using CellQuest software.
VOL. 79, 2005Mamu-A*01-ASSOCIATED PROTECTION AGAINST SHIV-89.6P4581
Statistical analysis. The exact Wilcoxon rank sum test was used to compare
CD4?T lymphocytes, plasma viral RNA levels, and Gag-specific ELISPOT and
tetramer responses between Mamu-A*01?and Mamu-A*01?rhesus monkeys in
the unvaccinated and vaccinated groups. Viral loads below 125 (the lower de-
tection limit of the assay) were treated as though they were 125. All tests were
two sided. The power statements for these tests were based on the observed
standard deviations and the fact that the asymptotic efficiency of the t test relative
to that of the Wilcoxon rank sum test is no greater than 1.16 for any continuous
distribution. There were no corrections for multiple comparisons. The compar-
ison of times to progression for control monkeys used the log rank statistic; the
power statement for this test is an approximation based on the assumption that
both distributions of times are exponential with a guarantee time of 100 days.
Plasma viral RNA levels, peripheral blood CD4?T-lympho-
cyte counts, and survival in unvaccinated Mamu-A*01?and
Mamu-A*01?rhesus monkeys following SHIV-89.6P infec-
tion. We first sought to determine whether expression of the
MHC class I allele Mamu-A*01 in immunologically naive rhe-
sus monkeys influenced viral replication and the rate of disease
progression following infection with SHIV-89.6P. A total of
11 Mamu-A*01?and 23 Mamu-A*01?rhesus monkeys that
served as control animals in two vaccine studies were evalu-
ated. These unvaccinated monkeys were challenged intrave-
nously with 50 MID50of SHIV-89.6P. Plasma viral RNA levels
peaked between days 14 and 16 after challenge for both the
Mamu-A*01?and Mamu-A*01?monkeys, with similar mean
log10values of 8.1 copies of viral RNA per ml measured in
each group of animals (Fig. 1, upper panels, left versus right).
Long-term containment of viral replication was evaluated in
these monkeys by measuring plasma viral RNA levels between
days 42 and 168 following challenge. A set point viral RNA
level for each monkey was calculated as the mean of these
values; data for each animal included at least seven time points
within this time interval. No significant difference in viral rep-
lication during this chronic phase of infection between these
groups of animals was detected, with mean log10set point
plasma viral RNA levels of 5.7 and 6.1 copies per ml measured
for Mamu-A*01?and Mamu-A*01?monkeys, respectively
(P ? 0.56).
Because SHIV-89.6P infection of rhesus monkeys causes a
dramatic and persistent decline in their peripheral blood
CD4?T lymphocyte levels, we assessed whether a difference in
CD4?T-lymphocyte depletion could be detected between the
Mamu-A*01?and Mamu-A*01?monkeys. Most monkeys in
both groups of animals exhibited a profound loss of peripheral
blood CD4?T lymphocytes within the first 21 days of infection
(Fig. 1, lower panels, left versus right). No significant differ-
ence in the percentages of peripheral blood CD4?T cells was
detected between the Mamu-A*01?and Mamu-A*01?mon-
keys between days 42 and 168 postchallenge (P ? 0.34). To-
gether with the viral load analysis, these results suggest that
expression of Mamu-A*01 in immunologically naive rhesus
monkeys does not affect acute or chronic SHIV-89.6P replica-
tion or the associated loss of CD4?T lymphocytes.
Expression of Mamu-A*01 also did not influence the rate of
disease progression in these animals following challenge.
Mamu-A*01?and Mamu-A*01?rhesus monkeys had similar
survival curves following SHIV-89.6P infection (Fig. 2). The
median times to death, 196 days for Mamu-A*01?monkeys
and 273 days for Mamu-A*01?monkeys, were not significantly
different (P ? 0.43, two-sided log rank test). By day 300 postin-
fection, only three Mamu-A*01?monkeys (27%) and eight
Mamu-A*01?monkeys (35%) remained alive. Thus, expres-
FIG. 1. Plasma viral RNA levels and peripheral blood CD4?T lymphocytes in unvaccinated Mamu-A*01?and Mamu-A*01?rhesus monkeys
following SHIV-89.6P infection. Plasma viral RNA levels (top panels) were measured using an ultrasensitive branched DNA amplification assay
with a detection limit of 125 copies/ml (dashed lines). Log10viral RNA copies/ml in plasma samples from individual monkeys are indicated. The
percentage of CD3?CD4?T lymphocytes in the peripheral blood of infected monkeys was assessed by cell staining and flow cytometric analysis
4582SEAMAN ET AL.J. VIROL.
sion of Mamu-A*01 in immunologically naive rhesus monkeys
does not appear to confer a survival advantage in the setting of
intravenous SHIV-89.6P infection.
Gag-specific cellular immune responses in unvaccinated,
SHIV-89.6P-infected Mamu-A*01?and Mamu-A*01?mon-
keys. Immunodominant Mamu-A*01-restricted CTL responses
specific for the SIV Gag-derived p11C epitope are associated
with decreased viral replication following SIV infection in rhe-
sus monkeys (20). We therefore examined whether the failure
to control viral replication in SHIV-89.6P-infected naive mon-
keys reflected an inability to generate the dominant Gag-spe-
cific CTL response in these Mamu-A*01?animals. The Gag-
specific cellular immune responses in the SHIV-89.6P-infected
Mamu-A*01?and Mamu-A*01?monkeys were assessed by
pooled peptide IFN-? ELISPOT assays using freshly isolated
PBL. The majority of the Mamu-A*01?and Mamu-A*01?
monkeys demonstrated low-frequency or undetectable Gag-
specific cellular responses on days 14 and 28 postinfection
(Fig. 3). These data indicate that both naive Mamu-A*01?and
Mamu-A*01?monkeys infected with SHIV-89.6P fail to de-
velop robust Gag-specific cellular immune responses.
Plasma viral RNA levels and peripheral blood CD4?T-
lymphocyte counts in vaccinated Mamu-A*01?and Mamu-
A*01?rhesus monkeys following SHIV-89.6P infection. We
next examined whether expression of Mamu-A*01 in previ-
ously vaccinated rhesus monkeys conferred clinical protection
following SHIV-89.6P challenge. Data on Mamu-A*01?and
Mamu-A*01?monkeys from two independent vaccine studies
were analyzed. In the first study, 12 Mamu-A*01?and 16
Mamu-A*01?monkeys received DNA prime and DNA or
recombinant poxvirus boost immunizations with vaccine vec-
tors encoding SIVmac239 Gag and HIV-1 89.6P Env immu-
nogens (25). At 18 weeks following the last immunization, all
monkeys received an intravenous inoculation of 50 MID50of
SHIV-89.6P, the same challenge dose used for unvaccinated
animals. Viral replication did not differ significantly between
the vaccinated Mamu-A*01?and Mamu-A*01?monkeys dur-
ing acute infection (Fig. 4A). Peak plasma viral loads were
detected on day 14 postchallenge in both groups of animals,
with mean log10values of 6.8 and 7.2 copies of viral RNA per
ml measured in the Mamu-A*01?and Mamu-A*01?monkeys,
respectively (P ? 0.73). Long-term containment of viral repli-
cation in these groups of monkeys was evaluated by measuring
plasma viral RNA levels between days 42 and 300 following
challenge. Mamu-A*01?monkeys exhibited a trend toward
lower set point plasma viral RNA levels during this time period
compared with Mamu-A*01?monkeys (P ? 0.12). This dif-
ference appeared to become more marked at the later time
points. All DNA prime- and DNA or recombinant poxvirus
FIG. 2. Survival curves of unvaccinated Mamu-A*01?and Mamu-
A*01?rhesus monkeys following intravenous infection with 50 MID50
FIG. 3. Cellular immune responses to SIV Gag in unvaccinated
Mamu-A*01?and Mamu-A*01?rhesus monkeys following SHIV-
89.6P infection. PBL were freshly isolated at days 14 and 28 postin-
fection and assessed for IFN-? ELISPOT responses following stimu-
lation with a peptide pool spanning the SIV Gag protein. Data are
presented as the number of SFC per 106PBL from individual monkeys,
with bars indicating the mean responses from 11 Mamu-A*01?(closed
symbols) and 23 Mamu-A*01?(open symbols) monkeys.
FIG. 4. Plasma viral RNA levels and peripheral blood CD4?T
lymphocytes in DNA prime- and DNA or recombinant poxvirus boost-
immunized Mamu-A*01?and Mamu-A*01?rhesus monkeys following
SHIV-89.6P challenge. Plasma viral RNA levels were measured using
an ultrasensitive branched DNA amplification assay with a detection
limit of 125 copies/ml (dashed line). The percentage of CD3?CD4?T
lymphocytes in the peripheral blood of infected monkeys was assessed
by cell staining and flow cytometric analysis. Data are presented as the
mean number of log10viral RNA copies ? SEM/ml (A) and the mean
percentage of CD4?PBL ? SEM (B) from 12 Mamu-A*01?(closed
symbols) and 16 Mamu-A*01?vaccinated (open symbols) monkeys.
VOL. 79, 2005Mamu-A*01-ASSOCIATED PROTECTION AGAINST SHIV-89.6P 4583
boost-vaccinated monkeys demonstrated blunting of periph-
eral blood CD4?T-lymphocyte loss compared to unvaccinated
control monkeys (Fig. 4B versus Fig. 1 [lower panels]). Levels
of CD4?T lymphocytes during chronic infection were higher
in Mamu-A*01?than in Mamu-A*01?monkeys, although this
difference did not approach statistical significance (P ? 0.50).
In the second vaccine study, 8 Mamu-A*01?and 16 Mamu-
A*01?monkeys received DNA prime/recombinant adenovirus
boost immunizations with vectors encoding SIVmac239 Gag-
Pol-Nef polyprotein and HIV-1 Env immunogens (26). At 16
weeks after the final immunization, all monkeys were chal-
lenged intravenously with 50 MID50of SHIV-89.6P. No
significant differences in peak plasma viral RNA levels were
detected between Mamu-A*01?and Mamu-A*01?monkeys
(Fig. 5A) (P ? 0.54). Set point plasma viral RNA levels be-
tween days 42 and 300 postchallenge, however, were signifi-
cantly lower in the Mamu-A*01?monkeys than in the Mamu-
A*01?monkeys (mean values of 3.3 and 4.8 log10copies per
ml, respectively; P ? 0.01). Furthermore, vaccinated Mamu-
A*01?monkeys demonstrated a trend toward greater preser-
vation of peripheral blood CD4?T lymphocytes during this
time period than did the vaccinated Mamu-A*01?monkeys
(Fig. 5B) (P ? 0.08). The data from these two studies therefore
suggest that in the setting of prior vaccination, Mamu-A*01?
monkeys demonstrate significantly better control of chronic
viral replication and a trend toward better preservation of
CD4?T lymphocytes than do Mamu-A*01?monkeys. Never-
theless, all experimentally vaccinated monkeys from these two
vaccine studies, Mamu-A*01?and Mamu-A*01?, remained
alive through the period of evaluation, 300 days postchallenge.
Thus, differences in the rates of disease progression in vacci-
nated, SHIV-89.6P-infected Mamu-A*01?and Mamu-A*01?
monkeys could not be determined.
Gag-specific cellular immune responses in Mamu-A*01?
and Mamu-A*01?monkeys following vaccination and SHIV-
89.6P infection. Since cellular immune responses contribute
to primate lentivirus containment, we sought to determine
whether differences in virus-specific CD8?T-lymphocyte re-
sponses might explain the improved control of SHIV-89.6P
replication in the vaccinated Mamu-A*01?monkeys. We first
examined whether the Mamu-A*01?monkeys demonstrated
higher-frequency Gag-specific CTL responses than the Mamu-
A*01?monkeys following immunization and/or challenge.
Freshly isolated PBL from monkeys receiving DNA prime and
DNA or recombinant poxvirus boost or DNA prime/rAd boost
vaccinations were assessed for Gag-specific cellular immune
responses by pooled peptide IFN-? ELISPOT assay. PBL of
both the Mamu-A*01?and Mamu-A*01?monkeys immunized
with plasmid DNA and recombinant poxvirus vectors demon-
strated robust Gag-specific cellular immune responses (Fig.
6A). In fact, no significant differences between these cohorts of
monkeys were observed in the numbers of spot-forming cells
(SFC) generated following either the DNA prime or recombi-
nant poxvirus boost immunizations (P ? 0.19 and 0.14, respec-
tively). Further, the magnitudes of the PBL Gag-specific SFC
FIG. 5. Plasma viral RNA levels and peripheral blood CD4?T
lymphocytes in DNA prime/rAd boost-immunized Mamu-A*01?and
Mamu-A*01?rhesus monkeys following SHIV-89.6P challenge. Plas-
ma viral RNA levels were measured using an ultrasensitive branched
DNA amplification assay with a detection limit of 125 copies/ml
(dashed line). The percentage of CD3?CD4?T lymphocytes in the
peripheral blood of infected monkeys was assessed by cell staining and
flow cytometric analysis. Data are presented as the number of mean
log10viral RNA copies ? SEM/ml (A) and the mean percentage of
CD4?PBL ? SEM (B) from 8 Mamu-A*01?(closed symbols) and 16
Mamu-A*01?vaccinated (open symbols) monkeys.
FIG. 6. Cellular immune responses to SIV Gag in Mamu-A*01?
and Mamu-A*01?rhesus monkeys following vaccination and SHIV-
89.6P challenge. Monkeys received DNA prime- and DNA or poxvirus
boost-immunizations (A) or DNA prime/rAd boost immunizations (B)
prior to challenge with SHIV-89.6P. PBL were isolated at the indicated
times following vaccination and challenge and assessed for IFN-?
ELISPOT responses following exposure to a peptide pool spanning the
SIV Gag protein. Data are presented as the mean number of SFC per
106PBL from each group of monkeys ? SEM.
4584SEAMAN ET AL.J. VIROL.
responses of these groups of monkeys were indistinguishable
on the day of SHIV-89.6 challenge (P ? 0.20).
Following challenge, however, PBL from the Mamu-A*01?
monkeys demonstrated higher-frequency Gag-specific cellular
immune responses than did PBL of the Mamu-A*01?mon-
keys. The mean SFC responses of PBL of the Mamu-A*01?
and Mamu-A*01?monkeys on day 14 postchallenge were
6,167 ? 792 (standard error of the mean [SEM]) and 3,443 ?
550, respectively (P ? 0.004); and on day 28 postchallenge
these immune responses were 3,496 ? 240 and 1,553 ? 327,
respectively (P ? 0.0003). Similar results were observed in
monkeys receiving DNA prime/rAd boost immunizations (Fig.
6B). The magnitudes of the peak Gag-specific SFC responses
were similar in Mamu-A*01?and Mamu-A*01?monkeys fol-
lowing both the DNA prime (P ? 0.13) and the rAd boost (P ?
0.08) immunizations; however, the frequency of Gag-specific
responses was higher in Mamu-A*01?monkeys than in Mamu-
A*01?monkeys on the day of challenge (P ? 0.003), 16 weeks
following the rAd boost. PBL of these Mamu-A*01?monkeys
further demonstrated higher-frequency Gag-specific cellular
immune responses immediately following SHIV-89.6P chal-
lenge, with mean SFC responses from Mamu-A*01?and Mamu-
A*01?monkeys measuring 4,331 ? 574 and 1,843 ? 417,
respectively, on day 14 (P ? 0.007), and 3,253 ? 285 and 1,709
? 220 on day 28 following challenge (P ? 0.002). Interestingly,
while PBL of both cohorts of vaccinated Mamu-A*01?mon-
keys exhibited higher-frequency Gag-specific cellular immune
responses than PBL of vaccinated Mamu-A*01?monkeys fol-
lowing SHIV-89.6P infection, this difference in cellular im-
mune responses did not extend to all viral antigens. In fact, cel-
lular immune responses to 89.6P Env were equivalent in PBL
of vaccinated Mamu-A*01?and Mamu-A*01?monkeys on
days 14 and 28 following challenge in both vaccine studies
We then assessed the extent to which the differences in
the magnitudes of the Gag-specific T-cell responses reflected
the contribution of the dominant p11C epitope-specific CTL
response. We first evaluated the contribution of p11C epitope-
specific CTL to the total Gag-specific cellular immune re-
sponse measured in individual DNA prime/rAd boost-im-
munized Mamu-A*01?monkeys on day 28 postchallenge. As
shown in Fig. 8 (left panel), SFC responses to the p11C epitope
peptide (hatched bars) were equivalent in magnitude to the
responses specific for the entire Gag peptide pool (solid bars)
for seven of eight monkeys. These data suggest that CTL spe-
cific for the p11C epitope dominate the Gag-specific immune
response in the Mamu-A*01?monkeys. For comparison, we
also measured Gag epitope-specific CTL responses for seven
monkeys in the Mamu-A*01?group that expressed the MHC
class I allele Mamu-A*02. The Gag-derived peptide p17G (also
referred to as GY9) has previously been described as a dom-
inant Mamu-A*02-restricted CTL epitope (28). As shown in
Fig. 8 (middle panel), SFC responses specific for the p17G
peptide (hatched bars) were equivalent in magnitude to re-
sponses specific for the entire Gag peptide pool (solid bars) for
all seven monkeys. Yet, despite the fact that p17G-specific
CTL are dominant Gag-epitope-specific CTL in Mamu-A*02?
rhesus monkeys, cellular immune responses to the Gag antigen
were significantly lower in PBL of Mamu-A*02?monkeys (P ?
0.004) and Mamu-A*01?/A*02?monkeys (P ? 0.006) (Fig. 8,
right panel) than in PBL of the Mamu-A*01?animals.
Tetramer staining analysis provided further evidence for the
contribution of the CD8?T-cell responses specific for the
p11C epitope to viral containment postchallenge in the Mamu-
A*01?monkeys. Tetramer staining was performed on PBL of
the vaccinated-challenged monkeys to determine the magni-
tudes of the p11C-specific CTL responses in the Mamu-A*01?
FIG. 7. Cellular immune responses to 89.6P Env in vaccinated
Mamu-A*01?and Mamu-A*01?rhesus monkeys following SHIV-
89.6P challenge. Monkeys received DNA prime- and DNA or recom-
binant poxvirus-boost immunizations (left panel) or DNA prime/rAd
boost immunizations (right panel) prior to challenge with SHIV-89.6P.
PBL were isolated on days 14 and 28 following challenge and assessed
for IFN-? ELISPOT responses following exposure to a peptide pool
spanning the HIV-1 89.6P Env protein. Data are presented as the mean
number of SFC per 106PBL from each group of monkeys ? SEM.
FIG. 8. Cellular immune responses to SIV Gag and Gag-derived epitope peptides in DNA prime/rAd boost-immunized Mamu-A*01?,
Mamu-A*02?, and Mamu-A*01?/A*02?monkeys following SHIV-89.6P challenge. PBL were isolated at day 28 postchallenge and assessed for
IFN-? ELISPOT responses following exposure to a peptide pool spanning the SIV Gag protein (all monkeys) or the Gag-derived dominant-epitope
peptide p11C (Mamu-A*01?monkeys) or p17G (Mamu-A*02?monkeys). Data are presented as the number of SFC per 106PBL from individual
VOL. 79, 2005Mamu-A*01-ASSOCIATED PROTECTION AGAINST SHIV-89.6P4585
monkeys and of the p17G-specific CTL responses in Mamu-
A*02?monkeys following both vaccination and challenge (Fig.
9). Mean responses to p11C and p17G were 6.7% ? 1.6% and
1.9% ? 0.7% of CD3?CD8?T lymphocytes, respectively,
following the rAd boost immunization (P ? 0.006), 27.3% ?
6.8% and 7.4% ? 1.7% on day 14 postchallenge (P ? 0.029),
and 16.5% ? 3.6% and 4.3% ? 1.2% on day 28 postchallenge
(P ? 0.009). Thus, the tetramer staining data indicated that the
p11C-specific CTL responses in PBL of the Mamu-A*01?
monkeys were significantly higher in magnitude than the
p17G-specific CTL responses in PBL of the Mamu-A*02?
monkeys, both postvaccination and postchallenge. Together,
these data demonstrate that vaccinated Mamu-A*01?monkeys
develop a higher-magnitude response to Gag following SHIV-
89.6P challenge than vaccinated Mamu-A*01?monkeys and
that this enhanced response reflects the contribution of a single
dominant Gag-epitope-specific CTL response.
While we have demonstrated an association between the
expression of the MHC class I molecule Mamu-A*01 and de-
layed disease progression in rhesus monkeys infected with
SHIV-89.P, this protective effect was apparent in the setting of
prior vaccination but not in that of immunologically naive
animals. In the previous studies demonstrating an association
between Mamu-A*01 expression and clinical protection in
monkeys, immunologically naive monkeys were experimentally
infected with the pathogenic virus SIVmac251, SIVsmE660, or
SIVmac239 (15, 16, 20, 21). The Mamu-A*01?monkeys dem-
onstrated lower set point viral loads, a greater preservation
of CD4?T lymphocytes, or increased survival compared to
Mamu-A*01?monkeys. Our inability to demonstrate signifi-
cant clinical differences between naive Mamu-A*01?and
Mamu-A*01?monkeys infected with SHIV-89.6P may be ex-
plained by the dramatic loss of CD4?T lymphocytes that is
seen within the first few weeks following SHIV-89.6P infection.
Studies of both humans and monkeys have demonstrated
that robust CD4?T-helper-cell responses generated early af-
ter infection can ameliorate disease progression, most likely
due to the ability of these cells to potentiate antiviral CD8?
T-cell and B-cell immunity (11, 12, 24). However, monkeys
infected with SHIV-89.6P have been shown to develop a rapid
and almost complete depletion of CXCR4-expressing naı ¨ve
and central memory CD4?T cells (19). Unvaccinated monkeys
that are infected with SHIV-89.6P may therefore lose the
CD4?T-cell help needed to mount efficient effector CTL re-
sponses, thereby eliminating the contribution of the Mamu-
A*01-associated CTL-associated protection. The inability of
unvaccinated Mamu-A*01?monkeys infected with SHIV-
89.6P to develop robust Gag-specific cellular immune re-
sponses is consistent with this hypothesis (Fig. 3). Whether
there are qualitative differences in the CTL populations gen-
erated in unvaccinated and vaccinated monkeys following
SHIV-89.6P infection remains to be elucidated.
A recent study demonstrated attenuated disease progression
in unvaccinated Mamu-A*01?monkeys infected with SHIV-
89.6P (29). Infected monkeys expressing Mamu-A*01 were re-
ported to develop lower viral loads in lymphoid tissues and
better preservation of lymph node architecture than monkeys
that were Mamu-A*01?. These particular parameters of dis-
ease were not assessed in the present report. While Mamu-
A*01?monkeys in that earlier study had longer survival times
than Mamu-A*01?monkeys, no significant differences in acute
or chronic plasma viral RNA levels or preservation of periph-
eral blood CD4?T lymphocytes were observed following in-
fection between these two groups of animals. The discrepancy
in the duration of survival of Mamu-A*01?monkeys between
the earlier and present study may reflect other contributing
Following SHIV-89.6P infection, robust cellular immune re-
sponses were observed for all monkeys that had previously
received experimental vaccines. The populations of virus-spe-
cific T cells that expanded in the vaccinated monkeys following
infection likely contributed to a partial containment of viral
replication and an associated blunting of the CD4?T-cell loss
during the first days after infection. Interestingly, the peak
Gag-specific cellular immune responses elicited in Mamu-
A*01?and Mamu-A*01?monkeys following both the DNA
prime and recombinant poxvirus or rAd boost immunizations
were of similar frequencies. However, in the DNA prime/rAd
boost-immunized monkeys, but not in the DNA prime/recom-
binant poxvirus-boosted monkeys, higher-frequency Gag-spe-
cific responses were detected in Mamu-A*01?than in Mamu-
A*01?monkeys on the day of challenge, 16 weeks following
the last immunization. Whether rAd and recombinant poxvirus
vectors differ in their abilities to generate long-lived CTL pop-
ulations when utilized as vaccine-boosting modalities warrants
evaluation. Following viral challenge, the Mamu-A*01?mon-
keys in both vaccine studies generated significantly higher-
frequency Gag-specific T-cell responses. Most or all of this
differential in T-cell responses could be attributed to p11C-
specific CTL. The fact that cellular immune responses to 89.6P
Env were of similar magnitudes in vaccinated Mamu-A*01?
and Mamu-A*01?monkeys following infection (Fig. 7) further
suggests that the dominant CTL response to the p11C epitope
provided the incremental protection observed in Mamu-A*01?
monkeys. Even the immunodominant Gag-specific CTL re-
sponses elicited in immunized Mamu-A*02?monkeys were
lower in frequency than the Mamu-A*01?-restricted p11C re-
FIG. 9. Tetramer staining analysis of CD8?T-lymphocyte re-
sponses to Gag-derived epitope peptides in Mamu-A*01?and Mamu-
A*02?monkeys following DNA prime/rAd boost immunizations and
SHIV-89.6P challenge. PBL were freshly isolated from Mamu-A*01?
and Mamu-A*02?monkeys at the indicated times following immuni-
zation and challenge, and epitope-specific CD8?T cells were detected
using Mamu-A*01/p11C or Mamu-A*02/p17G tetramers, respectively.
Data are presented as the percentages of gated CD3?CD8?PBL that
bound tetramer as measured by flow cytometry and are the means of
the results obtained with eight Mamu-A*01?or seven Mamu-A*02?
monkeys per group ? SEM.
4586SEAMAN ET AL.J. VIROL.
sponses following challenge. These data suggest that Mamu-
A*01?monkeys may have a significant advantage in generating
effective populations of T cells that can then expand rapidly
following reexposure to antigen.
We are grateful to Michelle Lifton, Darci Gorgone, Kristin Beaudry,
Kristi Martin, Margaret Beddall, Ayako Miura, Birgit Korioth-
Schmitz, Georgia Krivulka, and Faye Yu for excellent technical assis-
tance, Srini Rao, Jim Treece, Sharon Orndorff, and Debra Weiss for
management of nonhuman primate studies, Vi Dang and Alida Ault
for conduct of animal studies, and Nancy Miller for helpful conversa-
This work was supported by National Institutes of Health grant
AI30033 and National Cancer Institute/SAIC-Frederick contract
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