JOURNAL OF VIROLOGY, Dec. 2003, p. 13348–13360
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 24
Multispecific Vaccine-Induced Mucosal Cytotoxic T Lymphocytes
Reduce Acute-Phase Viral Replication but Fail in Long-Term
Control of Simian Immunodeficiency Virus SIVmac239
Thorsten U. Vogel,1† Matthew R. Reynolds,1,2Deborah H. Fuller,3Kathy Vielhuber,1Tim Shipley,3
James T. Fuller,3Kevin J. Kunstman,4Gerd Sutter,5Marta L. Marthas,6Volker Erfle,5
Steven M. Wolinsky,4Chenxi Wang,7,8David B. Allison,7,8Erling W. Rud,9Nancy Wilson,1
David Montefiori,10John D. Altman,11and David I. Watkins1,2*
Wisconsin Primate Research Center1and Department of Pathology and Laboratory Medicine,2University of Wisconsin, Madison,
Wisconsin 53715; PowderJect Vaccines, Madison, Wisconsin 537113; Northwestern University Medical School, Chicago, Illinois
606114; GSF–Institute for Molecular Virology, Munich, Germany5; California National Primate Research Center, University of
California, Davis, California 956166; Section on Statistical Genetics, Department of Biostatistics,7and Clinical Nutrition
Research Center, Department of Nutrition Sciences,8University of Alabama at Birmingham, Birmingham, Alabama
35294; National Laboratory for HIV Pathogenesis, Health Canada, Ottawa, Ontario K1A 0L2, Canada9; Center for
AIDS Research, Department of Surgery, Duke University Medical Center, Durham, North Carolina 2771010;
and Vaccine Research Center and Department of Microbiology and Immunology, Emory University
School of Medicine, Atlanta, Georgia 3031111
Received 3 June 2003/Accepted 8 September 2003
Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodefi-
ciency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte
(CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human
immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course
of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is
sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently,
SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To
ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on
the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01?macaques with constructs
encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA
prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-
specific CTL and CD4?helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood
mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL.
At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01?controls were challenged
intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase
viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The
present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve
a modicum of control over early viral replication but are unable to control chronic-phase viral replication after
a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.
Recent studies have demonstrated protection from disease
progression, offering hope that cytotoxic-T-lymphocyte (CTL)-
based vaccines might ameliorate the course of human immu-
nodeficiency virus (HIV) disease (6, 8, 50). After infection in
these studies, some macaques had strong anamnestic CTL
responses associated with reduced viral loads. Each of these
studies used SHIV-89.6P, a chimeric simian immunodeficiency
virus (SIV) expressing env, tat, rev, and vpu genes of HIV type
1 (HIV-1) isolate 89.6, as the challenge virus (41). However,
unlike most primary HIV strains, simian/human immunodefi-
ciency virus SHIV-89.6P causes acute CD4?-T-lymphocyte
loss and is sensitive to neutralizing antibodies (35, 41). It is
difficult, therefore, to extrapolate these studies to HIV infec-
tion in humans. Protection from the acute depletion of CD4?
T lymphocytes caused by SHIV-89.6P may allow for the devel-
opment of an effective antibody response to arise that is capa-
ble of controlling SHIV-89.6P replication. Conversely, the mo-
lecular clone SIVmac239 is difficult to neutralize and infected
macaques have a protracted decline in CD4?T lymphocytes.
Mucosal tissues are an active site of replication for both HIV
and SIV (48, 51). Ideally, vaccine-induced CTL could prevent
the virus from spreading systemically by rapidly eliminating
infected cells at the site of exposure. Studies in murine models
suggest that mucosal, but not systemic, CTL are capable of
protecting against mucosally transmitted viruses (9, 44). Since
the majority of HIV infections occur across mucosal surfaces,
targeting of immune responses to the mucosa may provide
immediate effector cells at the site of infection. Furthermore,
the importance of mucosal CTL may not only be spatial but
also functional. In mice, vesicular stomatitis virus-specific
* Corresponding author. Mailing address: Wisconsin Primate Re-
search Center, 1220 Capital Ct., Madison, WI 53715. Phone: (608)
265-3380. Fax: (608) 265-8084. E-mail: firstname.lastname@example.org.
† Present address: Aventis Pasteur, Toronto, Ontario M2R 3T4,
memory T cells derived from nonlymphatic tissue have been
shown to be more directly lytic than corresponding cells de-
rived from lymphatic tissues (33). This suggests that HIV-
specific memory T-lymphocytes, residing in nonlymphatic tis-
sues (small intestine, vagina, colon, etc.), may be able to react
immediately to HIV infection.
In a previous study we used a DNA prime/recombinant
modified vaccinia virus Ankara (rMVA) vaccination regimen
to induce a massive CTL response to a single Mamu-A*01-
restricted epitope, Gag181-189CM9 (5). When vaccinated
animals were challenged with SIVmac239, we observed no
amelioration of disease. Subsequently, we described a Mamu-
A*01-restricted CTL epitope in Tat that escaped early in in-
fection, suggesting that this epitope was under selective pres-
sure (3, 37). Since the Gag181-189CM9 epitope escapes only
intermittently in chronic infection (1, 37), we hypothesized that
this epitope might be under less selective pressure from CTLs
and therefore a less attractive vaccine target than the
Tat28-35SL8 epitope. We, therefore, designed a similar vaccine
that induced Tat-specific CTLs and determined whether they
could control acute virus replication (2). Despite a high-level
anamnestic CTL response, the vaccinated macaques were also
unable to control replication of SIVmac239. We then designed
another vaccine, by using constructs encoding all proteins of
SIV (including Env), which did result in lower viral replication
in the acute phase (23). Unfortunately, we could not exclude
the possibility that control was due to vaccine-induced Env-
specific antibody, even though no SIVmac239 neutralizing an-
tibodies were detected either before or after challenge.
We tested here whether CTLs directed against multiple
epitopes could control replication of SIVmac239 in the ab-
sence of Env-specific antibody. We also sought to determine
whether a vaccine regimen targeting CTLs to both systemic
and mucosal tissues would be more effective against viral chal-
lenge than vaccines now in use. Therefore, we vaccinated three
Mamu-A*01?rhesus macaques with the Mamu-A*01-re-
stricted CTL epitopes Gag181-189CM9 and Tat28-35SL8, along
with full-length SIV Tat, Rev, and Nef, by using a DNA prime/
rMVA boost regimen. A recent study showed that the intra-
venous (i.v.) administration of vaccinia virus could induce long-
lived memory T cells in the mucosa (32). For this reason the
final rMVA boost was delivered i.v.
MATERIALS AND METHODS
Animals. Rhesus macaques (Macaca mulatta) were maintained in accordance
with the NIH Guide to the Care and Use of Laboratory Animals, and under the
approval of the University of Wisconsin and the University of California Re-
search Animal Resource Center (RARC) review committees.
Peptides. Overlapping peptides (20-mers, 15-mers, 10-mers, 9-mers, and
8-mers) were synthesized by Chiron (Raleigh, N.C.), the Natural and Medical
Science Institute (University of Tuebingen, Germany), or the Biotechnology
Center (University of Wisconsin–Madison) based on SIVmac239 protein se-
quences, with the exception of Pol peptides, which corresponded to the
SIVmac251 sequence. Lyophilized peptides were resuspended in phosphate-
buffered saline (PBS) with 10% dimethyl sulfoxide (Sigma Chemical Co., St.
Louis, Mo.). Consecutive 20-mer, 15-mer, and 9-mer peptides overlap by 10, 11,
or 8 amino acids, respectively. Pools of peptides contained 10 peptides at a final
concentration of 1 mg/ml per peptide.
PBMC. Peripheral blood mononuclear cells (PBMC) were separated from
whole heparinized blood by Ficoll-diatrizoate (Histopaque; Sigma) density gra-
dient centrifugation and cultured according to methods described previously
B-LCL lines. Rhesus monkey B-lymphoblastoid cell lines (B-LCL) were gen-
erated as described previously (52, 53) by incubating PBMC with herpesvirus
papio produced by S594 cells.
Generation of chimeric HBcAg-CTL epitope expressing DNA vaccine. The
hepatitis B virus core antigen (HBcAg) carrier expression vector pHBc expresses
HBcAg under the control of the cytomegalovirus (CMV) immediate-early pro-
moter (PJV 7198; PowderJect Vaccines, Inc., Madison, Wis.). It contains a
unique Bsp120I restriction site within the immunodominant loop of HBcAg
between amino acids 80 and 81 and a unique NotI restriction site at the 3? end
of the HBcAg gene, facilitating the insertion of epitopes at either site (27). To
construct chimeric HBcAg-epitope DNA vaccines, pHBc was digested with ei-
ther Bsp120I or NotI. Oligonucleotides encoding Bsp120I- or NotI-flanked,
codon-optimized SIV CTL epitopes were synthesized, annealed, and ligated into
pHBc at the immunodominant region or carboxy terminus, respectively, of
HBcAg. Clones containing inserts were identified by PCR as described previ-
ously (27) and sequenced to confirm insertion of the correct coding sequences
Generation of the PJV7343, a SIVmacC8 Nef expressing DNA vaccine. The
coding sequence for SIV Nef was amplified by PCR with the plasmid pNef C8
derived from the C8 isolate of SIVmac32H (a SIVmac251 derivative) (46) as a
template with primers JF121 (5?-GGA AAG CTT GCA ATC ATG GGT GGA
GCT ATT TCC AGG-3?) and JF124 (5?-GGT GGG CCC TCA GCG AGT TTC
CTT CTT GTC AG-3?) by a standard PCR methodology (1? PCR core buffer
with 15 mM MgCl2[Promega, Madison, Wis.], 0.4 ?M concentrations of primers,
200 ?M concentrations of deoxynucleoside triphosphates, 2.5 U of Taq polymer-
ase [Promega], 1.0 ng of template DNA, water to 100 ?l, and a mineral oil
overlay). After phenol-chloroform extraction and ethanol precipitation, the PCR
product was resuspended in Tris-EDTA buffer and cut with Hind3 and Bsp120
I to generate an insert fragment. A vector fragment was prepared by removing
the HBcAg coding region from plasmid pHBcAg (27) by cutting with HindIII
and NotI. The two fragments were ligated together, resulting in PJV7343. The
Nef insert in PJV7343 was sequenced, and no changes from the expected se-
quence were discovered.
Generation of the SIV Tat-expressing DNA vaccine. Two PCRs were per-
formed to create an intronless Tat coding fragment. PJV7135 (PowderJect Vac-
cines), a plasmid containing the SIV-17E-Fred genome served as the template
and the Tat encoding regions were amplified with primers JF35 (5?-GCG CTA
GCG AGA CAC CCT TGA GGG AG-3?) and JF37 (5?-CAA ACA ACA GAC
CCA TAT CCA ACA GGA C-3?) and with primers JF36 (5?-ATG GGT CTG
TTG TTT GAT GCA GAA GAT G-3?) and JF38 (5?-GCG GAT CCG TCT
ATC TGC CAA GGC CAG GAG C-3) by using standard PCR conditions. The
thermocycler conditions included an initial denaturation step at 95°C for 4 min,
followed by 30 cycles of a 1-min denaturation at 95°C, 1 min 15 s of annealing at
55°C, and a 1-min extension at 72°C. After a final 10-min extension step at 72°C,
the reactions were stored at 4°C. The two PCR products were electrophoresed on
a 1% agarose gel, stained with ethidium bromide, excised from the gel, and
soaked for 30 min at 65°C in 100 ?l of water to elute the PCR fragments. One
microliter of each gel eluate was used in a standard PCR with primers JF35and
JF38 to amplify the complete Tat coding sequence. The resulting PCR product
was purified and cut with NheI and BamHI for fragment insertion. A vector
fragment was prepared by removing the HBcAg from a signal peptide-less
version of plasmid pWRG7063 (27) by cutting with NheI and BamHI. The insert
and vector were then ligated, resulting in PJV7271. The Tat insert in PJV7271
was sequenced, and one change (tyrosine to serine) from the expected sequence
at position 44 was discovered.
DNA/rMVA vaccinations. Animals were immunized six times with DNA by
using the PowderJectXR1 device (PowderJect Vaccines). The first three DNA
immunizations were given epidermally (eight sites) at intervals of 4 to 9 weeks;
after a 7- to 14-week rest period, another three DNA immunizations were given
epidermally (eight sites) and orally (four sites into the cheek pouch and four sites
into the tongue) at intervals of 4 weeks. For the first three immunizations two
plasmid vectors expressing SIV Nef (pJV7343; see above) and HBcAg with the
CTL epitope Gag181-189CM9 (4) incorporated into the antigenic loop (see above)
were used. For the next set of three DNA immunizations, an additional three
vectors were used, expressing SIV Tat (pSIVTat; see above), SIV Rev (pSIVrev;
see reference 19), and HBcAg with the CTL epitope Tat28-35SL8 (3) incorpo-
rated into its antigenic loop (see above). Equal amounts of each plasmid DNA
were precipitated onto 1- to 3-?m gold particles (Degussa, Plainfield, N.J.) in the
presence of 0.1 M spermidine (Sigma) and 2.5 M CaCl2(Fujisawa, Inc., Melrose
Park, Ill.) at a rate of 4 ?g of DNA per mg of gold. One milligram of gold was
delivered per site.
Generation and inoculation of rMVA vector vaccines. rMVA constructs used
in the present study separately express the tat, rev, or nef coding sequences of the
VOL. 77, 2003CTLs REDUCE ACUTE-PHASE VIRAL REPLICATION 13349
SIVmac32H J5 clone (46) under the transcriptional control of the vaccinia virus
early/late promoter P7.5. We also used an rMVA virus expressing the CTL
epitope Gag181-189CM9 (4) as a minigene under the control of the P7.5 pro-
moter. To generate vaccine preparations, recombinant and nonrecombinant
MVA were amplified on chicken embryo fibroblast (CEF) cells derived from
embryonated eggs of a specific-pathogen-free stock. CEF were grown in minimal
essential medium (Biochrom, Berlin, Germany) supplemented with 10% fetal
bovine serum (Biochrom) and maintained in a humidified air–5% CO2atmo-
sphere at 37°C. Viruses were purified by ultracentrifugation through a cushion of
36% (wt/vol) sucrose in 10 mM Tris-Cl (pH 8.0) and reconstituted in PBS, and
titers were determined by immunostaining of virus-infected cell foci on CEF
monolayers by using vaccinia virus-specific rabbit polyclonal antibody (Biogen-
esis, Ltd., Poole, United Kingdom). Virus preparations were divided into ali-
quots that contained 5 ? 108infectious units/ml and stored at ?70°C. The vector
vaccine preparations were tested in vitro for their capacity to synthesize SIV
target antigens by Western blot analyses for Rev and Nef proteins, and Tat
production was confirmed by assaying the transcriptional activation of HIV-long-
terminal-repeat-controlled luciferase reporter gene expression (data not shown).
About 14 to 28 weeks after the last DNA vaccination, all animals of the vaccine
group were inoculated with rMVA vaccines encoding SIVmacJ5 (46) Nef, Rev,
and Tat and with rMVA encoding the CTL epitope Gag181-189CM9 delivered
intradermally (i.d.) and intranasally (i.n.). The animals received 108infectious
units of each rMVA vector vaccine. Control animals received equal amounts of
nonrecombinant MVA. After a 33- to 35-week rest period, all animals were
inoculated a second time with the same rMVA, but this time they were inocu-
lated i.v. No side effects or lesions were found associated with the inoculations.
Peptide-specific T-cell lines. Peptide-specific CD8?- and CD4?-T-cell lines
were generated by using previously described methods (53). Briefly, at day 0
fresh PBMC were in vitro stimulated with peptide-pulsed, autologous B-LCL as
stimulator cells. At day 7, CD8??cells and CD4?cells were separated by using
the Miltenyi Biotec MiniMACS system. The separated CD8??and CD4?cells
were again in vitro stimulated with peptide-pulsed, autologous B-LCL as stim-
ulator cells. After a total of 14 days of in vitro stimulation the cells were used as
effectors in intracellular cytokine staining (ICS) assays to test for the peptide-
specific release of gamma interferon (IFN-?).
ICS of fresh PBMC. ICS assays were performed as previously described (23).
Between 5 ? 105and 1 ? 106PBMC were incubated with either staphylococcal
enterotoxin B (10 ?g/ml; Sigma) as a positive control, pools of 10 15-mer and
20-mer peptides together, or individual peptides at a concentration of 5 ?g/ml,
along with 0.5 ?g of anti-CD28 (clone L293; BD Biosciences, San Diego, Calif.)
and 0.5 ?g of anti-CD49d (clone 9F10; BD Pharmingen) in a total volume of 200
?l of R-10 (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-
glutamine, 25 mM HEPES buffer, 25 ?M 2-mercaptoethanol, 50 ?g of strepto-
mycin/ml, and 50 U of penicillin/ml). Anti-CD28 and anti-CD49d antibodies
were added to provide optimal costimulation (38). After 1.5 h at 37°C, 10 ?g of
Brefeldin A (Sigma)/ml was added to inhibit secretion of cytokines, and the cells
were further incubated for 5 h at 37°C. Cells were washed twice with 1 ml of
fluorescence-activated cell sorting (FACS) buffer (PBS plus 2% fetal calf serum)
and then stained with 6 ?l of CD8?-PerCP (clone SK1; Becton Dickinson) and
4 ?l of CD4-allophycocyanin (APC) (clone SK3, Becton Dickinson) in 100 ?l of
FACS buffer for 40 min. After two washes with 1 ml of FACS buffer, the cells
were fixed with 2% paraformaldehyde (PFA)–PBS solution overnight at 4°C. The
cells were then washed once with FACS buffer, treated with permeabilization
buffer (0.1% saponin in FACS buffer) for 10 min at room temperature, washed
once more with 0.1% saponin buffer, and resuspended in 100 ?l of 0.1% saponin
buffer. Then, 1 ?l of anti-human IFN-?–fluorescein isothiocyanate (FITC)
monoclonal antibody (clone 4S.B3; Pharmingen) and 1 ?l of anti-human tumor
necrosis factor alpha-phycoerythrin (PE) monoclonal antibody (clone MAb11;
Pharmingen) were added. After 50 min of incubation at room temperature, the
cells were washed two times with 0.1% saponin buffer, followed by a 10-min
incubation before the last spin, and then fixed with 2% PFA-PBS. A total of
100,000 to 200,000 lymphocyte-gated events were acquired on a FACSCalibur
31107, 31157 & 30977†
17 31 35 39
CM9, Nef (e.d.)
Nef, Rev, Tat
CM9, Nef (e.d.)
Nef, Rev, Tat
23 27 31
31689 & 31821
FIG. 1. Immunization schedule. e.d., epidermal. †, Animal euthanized 4 weeks after the first rMVA boost.
13350 VOGEL ET AL.J. VIROL.
flow cytometer (Becton Dickinson) and analyzed by using FlowJo software
(Treestar). The background level of IFN-? staining in PBMC (induced by the
control influenza peptide SNEGSYFFG) varied from animal to animal but was
typically ?0.05% in CD8?lymphocytes and ?0.02% in CD4?lymphocytes. Only
samples displaying IFN-? staining at least twice that of the background or in
which there was a distinct population of IFN-? (bright)-positive cells (also pos-
itive for TNF-?) were considered positive. All values are reported after subtrac-
tion of the background level staining.
ICS with T-cell lines for fine mapping. When T-cell lines were analyzed by
ICS, the method described above (for fresh PBMC) was modified so that 105
B-LCL were used instead of anti-CD28 and anti-CD49d. The background level
of ICS in T-cell lines (induced by the control influenza peptide SNEGSYFFG)
was usually ?0.5% and was subtracted from all values.
Challenge with molecularly cloned SIVmac239/nef-open. At 17 weeks after the
last rMVA boost, three vaccinated animals and three controls were challenged
intrarectally (i.r.) with a molecularly cloned virus, SIVmac239/nef-open (40),
with a dose of approximately 10 i.r. monkey infectious doses (MID50) (36), as
described previously (23).
Viral load analysis. Viral RNA from SIV was quantitated by real-time PCR by
using the TaqMan assay kit (Perkin-Elmer Applied Biosystems, Foster City,
Calif.) and evaluated on an ABI Prism 7000 (Perkin-Elmer Applied Biosystems,
Foster City, CA) apparatus. Primer and probe sequences were as follows: for-
ward primer SIV-61F, 5?-CCACCTACCATTAAGCCCGA-3?; reverse primer
SIV-143R, 5?-CTGGCACTACTTCTGCTCCAAA-3?; and probe SIV-84T
(FAM reporter, TAMRA quencher), 5?-CATTAAATGCCTGGGTAAAATTG
ATAGAGGA(GA)AAGAA-3?. The reaction mixture contained 1? TaqMan
FIG. 2. Frequencies of CD8 and CD4 responses after the first rMVA boost in fresh PBMC as determined by ICS and tetramer staining. Fresh
PBMC were tested for SIV-specific cellular responses by using previously mapped peptides or peptide pools as stimuli in ICS 1 week after
administration of rMVA (two left columns) and all individual peptides contained in positive pools or previously mapped epitopes (right column)
at week 2 after administration of rMVA. In some cases, two overlapping peptides were included and are reported as the combination of the two
peptides rather than as two independent peptides. In addition, we determined the frequency of Mamu-A*01/CM9 and Mamu-A*01/SL8
tetramer-positive cells (bottom panels). Animal 31157 is shown as an example. All animals were analyzed by this method.
VOL. 77, 2003 CTLs REDUCE ACUTE-PHASE VIRAL REPLICATION 13351
EZ buffer, 3 mM magnesium acetate, 1.2 mM concentrations of deoxynucleoside
triphosphates, 100 nM SIV-84T probe, 400 nM final forward primer, 800 nM
final reverse primer, 2.5 U of rTth, and 2 ?l of RNA sample or RNA standard.
Cycling conditions were as follows: 50°C for 2 min, 60°C for 30 min, and 95°C for
5 min, followed by 40 cycles of 95°C 15 s, 60°C for 1 min, and 25°C for 2 min. The
data were collected during the extension phase only.
Statistical analysis. Viral load differences between groups were tested for
statistical significance by using Student t tests after log transformation of the data
to improve normality and homoscedasticity. In addition, Levene’s test for ho-
moscedasticity was conducted and, if differences were found to be significant, the
Welch correction for unequal variances was used. Finally, to further examine the
robustness of the results, a nonparametric test, the Mann-Whitney U test, was
performed. The P values for the nonparametric tests were calculated by exact
methods. All of the P values are two tailed.
Neutralizing antibody assays. Neutralization of a T-cell line-adapted stock of
SIVmac251 or molecularly cloned SIVmac239/nef-open was measured in
CEMx174 cells as described previously (34). Briefly, titers of neutralizing anti-
bodies in this assay are reported as the reciprocal plasma dilution at which 50%
of cells were protected from virus-induced killing as measured by neutral red
uptake. The assay stock of SIVmac251 in this case was generated in H9 cells and
is highly sensitive to neutralization. Neutralization of molecularly cloned
SIVmac239/nef-open was also measured in human PBMC as a reduction in p27
Gag antigen synthesis (34). The assay stock of SIVmac239/nef-open was gener-
ated in human PBMC and was derived from the animal challenge stock.
Lymphocyte isolation from pinch biopsies. Six to eight pinch biopsies approx-
imately 2 by 2 by 2 mm in size were collected from the sigmoid colon by using a
Fujinon FG-100PE pediatric gastroscope. The biopsies were incubated three
successive times in an orbital shaker at 37°C for 30 min in R10 containing 15 ?g
of collagenase type II (Sigma)/ml. The supernatant after each incubation period
was collected and pooled. Lymphocytes were isolated by overlaying the collected
cells on an isotonic Percoll (Amersham-Pharmacia, Piscataway, N.J.) gradient
(40% layered over 100%) and centrifuging them for 30 min at 800 rpm. Lym-
phocytes were collected from the 40%-100% Percoll interface and washed with
Tetramer staining. We used a previously described method (23) to stain
lymphocytes. Briefly, 5 ? 105to 1 ? 106fresh, unstimulated lymphocytes were
suspended in a 100-?l volume of FACS buffer. The cells were stained in the dark
for 40 min at room temperature with either the Mamu-A*01/CM9 or Mamu-
A*01/SL8 tetramer labeled with PE or APC (5 ?g/ml), anti-human CD3ε-FITC
(SP34; Pharmingen), and anti-CD8?-PerCP (clone SK1; Becton Dickinson). In
certain cases tetramer-positive cells were also phenotyped for the presence of
mucosal homing and retention markers by staining them with a mixture of
APC-labeled tetramers, CD8?-PerCP, and antibodies to ?4?7(PE labeled; Mil-
lenium Pharmaceuticals) and ?E?7(CD103 [Coulter Immunotech], FITC la-
beled). The cells were then washed two times with 1 ml of FACS buffer and fixed
by adding 2% PFA-PBS solution. Sample data were acquired on a FACSCalibur
instrument and analyzed by using CellQuest software (Becton Dickinson). Back-
ground tetramer staining of fresh, unstimulated PBMC from naive Mamu-A*01-
positive animals was routinely less than 0.08%.
Immunization. Our DNA/rMVA vaccine regimen was de-
signed to induce systemic and mucosal CTL against multiple
epitopes in the absence of Env-specific antibodies (Fig. 1).
Three Mamu-A*01?rhesus macaques were immunized with
DNA encoding SIV Tat, Rev, Nef, and two immunodominant
Mamu-A*01restricted epitopes,Gag181-189CM9 and
FIG. 3. Tetramer-positive lymphocytes in the peripheral blood lack mucosal surface and retention markers after rMVA boost 1 (given i.d. and
i.n.). Fresh PBMC obtained 1 week after the first rMVA boost were stained with anti-CD8, anti-?4?7, and anti-?E?7antibodies and Mamu-A*01/
CM9 tetramer. Only the results for CD8?tetramer-positive cells are shown.
TABLE 1. Frequencies of SIV-specific CD8 and CD4 responses 1
week after rMVA boost 1 (i.d. and i.n.) as determined by ICS
Frequency (%) of:
4.2 0.14 0.46
TABLE 2. Tetramer staining in PBMC and lymph nodes in animal
30977 3 weeks after rMVA boost 1 (i.d. and i.n.)
aLN, lymph nodes.
bPercentage of CD8?lymphocytes.
13352VOGEL ET AL.J. VIROL.
Tat28-35SL8. A fourth animal started the vaccination regimen
but was euthanized prior to completion of the study due to
unrelated health problems. At 2 weeks after the first DNA
immunization, we detected Mamu-A*01/CM9 tetramer-posi-
tive cells in fresh PBMC in all four macaques at frequencies
between 0.12 and 0.75% of the CD3?CD8?lymphocytes.
After the DNA immunizations, both CTL and helper T-lym-
phocyte (HTL) responses were detected in each animal by ICS
after in vitro stimulation. The first rMVA immunization was
administered both i.n. and i.d. Virus-specific CD8?lympho-
cytes were clearly detectable in PBMC after the boost (Fig. 2
and Table 1).
We also assayed vaccine-induced CD8?T cells for expres-
sion of mucosal homing and retention markers. Effector and
memory lymphocytes acquire “tissue-specific” adhesion and
chemoattractant receptors based on their site of primary
activation (13). This selective tissue tropism allows lympho-
cytes to maximize their chances of reencountering their spe-
cific antigen (12). ?4?7and ?E(CD103)?7are involved in
lymphocyte homing to and retention within the gut. ?4?7is
upregulated on lymphocytes primed in mucosal tissues and
signal cells that will ultimately return to the gastrointestinal
tract (13, 54), whereas ?E?7retains intraepithelial lympho-
cytes (IEL) within the mucosal tissue by binding to its li-
gand, E-cadherin, on the surface of epithelial cells (14, 22).
After the DNA immunizations, only a small subset of tet-
ramer-positive CD8?lymphocytes expressed ?4?7or ?E?7
(data not shown). Indeed, the rationale for delivering part
of the first rMVA boost i.n. was to stimulate mucosal CTLs.
Surprisingly, 1 week after the boost ?E?7expression was not
detectable and ?4?7was only detectable on a small fraction
of tetramer-positive lymphocytes in the blood (Fig. 3). In
FIG. 4. Tetramer-positive lymphocytes in the peripheral blood are detectable 31 weeks after rMVA boost 1 (given i.d. and i.n.). Fresh PBMC
obtained 30 weeks after the first rMVA boost were stained with anti-CD3, anti-CD8, Mamu-A*01/CM9 tetramer (A) and Mamu-A*01/SL8
tetramer (B). The frequencies are listed as the percentage of tetramer staining in the CD3?CD8?lymphocyte population.
VOL. 77, 2003 CTLs REDUCE ACUTE-PHASE VIRAL REPLICATION13353
animal 30977, euthanized 3 weeks after the first rMVA
boost, Mamu-A*01/CM9 and Mamu-A*01/SL8 tetramer-
positive cells were detected in lymph nodes throughout the
body (Table 2). The vaccine-induced CTL responses were
long-lived in the remaining three vaccinees. Mamu-A*01/
CM9 and Mamu-A*01/SL8 tetramer-positive cells could be
detected in PBMC 31 weeks after boosting with rMVA (Fig.
4), although the expression of mucosal homing and reten-
tion markers remained low (data not shown).
Based on recently published data demonstrating that i.v.
administration of vaccinia induced a sustained mucosal re-
sponse (32), we boosted the animals once more i.v. with
rMVA. In contrast to previous studies (5, 21), in which the
second rMVA boost, given via the same route as the first, did
not increase the frequency of antigen-specific lymphocytes
over the initial rMVA boost, the i.v. rMVA boost resulted in
very high levels of tetramer-positive lymphocytes (Fig. 5).
Mamu-A*01/CM9-specific CTLs reached levels of between 7
and 17% of all CD8?T lymphocytes (Fig. 5A). There was also
a boosting of the Mamu-A*01/SL8-specific response (Fig. 5B).
Other previously defined CD8?- and CD4?-T-lymphocyte re-
sponses to SIV were also detectable by ICS (Table 3). Impor-
tantly, we were now able to detect tetramer-positive cells ex-
pressing the mucosal homing and retention markers ?4?7and
FIG. 5. Frequencies of tetramer-positive lymphocytes in the peripheral blood after rMVA boost 2 (i.v.). Fresh PBMC obtained at different time
points after the second rMVA boost (i.v.) were stained with anti-CD3, anti-CD8, and Mamu-A*01/CM9 tetramers (A) or Mamu-A*01/SL8
tetramers (B). Percentage of tetramer-positive cells in the CD3?CD8?lymphocyte population. All control animals were negative for tetramer
staining (less than the background [i.e., 0.08%]).
TABLE 3. Frequencies of SIV-specific CD8 and CD4 responses
after rMVA boost 2 (i.v.) as determined by ICS.
Frequency (%) of CD8?lymphocytes
13354 VOGEL ET AL. J. VIROL.
?E?7(Fig. 6A). Virus-specific T cells expressing these markers
were also detectable on lymphocytes isolated from pinch biop-
sies taken from the sigmoid colon (Fig. 6B). The i.v. adminis-
tration of rMVA induced sustained, virus-specific CTL re-
sponses in both the blood and the sigmoid colon at 10 weeks
postboost (Fig. 7).
SIVmac239. To determine whether these long-lived cellular
immune responses could control SIVmac239 replication, we
challenged the three vaccinated animals, along with three na-
ive Mamu-A*01?controls, i.r. with 10 MID50SIVmac239 at 16
weeks after the final rMVA boost. The vaccinated animals
made robust anamnestic immune responses. In animal 31107
the Mamu-A*01/CM9-specific CTLs attained levels of 18.1%
of CD8?lymphocytes in the PBMC by 2 weeks postchallenge
(Fig. 8). In comparison, the highest Mamu-A*01/CM9 tet-
mucosal challenge with
ramer levels observed in any of the controls was 1.25%. More-
over, although Mamu-A*01/SL8-specific CTL levels were
modest after the final rMVA boost in comparison to Mamu-
A*01/CM9-specific CTL, in two of three vaccinated animals at
2 weeks postchallenge, Mamu-A*01/SL8 tetramer-positive
lymphocytes were present at a higher frequency than were
Mamu-A*01/CM9 tetramer-positive lymphocytes (Fig. 8A).
ICS assays with the corresponding peptides, Gag181-189CM9
and Tat28-35SL8, showed comparable, yet somewhat lower, fre-
quencies of CD8?lymphocytes than were observed by tet-
ramer staining (compare Fig. 8A and B). ICS also showed that,
in addition to Gag181-189CM9 and Tat28-35SL8, other regions of
Tat, Rev, and Nef that were recognized after immunization
were still recognized 2 weeks postchallenge, including several
CD4?-T-cell responses (data not shown).
FIG. 6. Tetramer-positive lymphocytes after rMVA boost 2 (i.v.) express mucosal homing and retention markers. Fresh PBMC (A) or IEL/LPL
obtained from intestinal punch biopsies (B) at 1 week after the second rMVA boost (i.v.) were stained with anti-CD8, Mamu-A*01/CM9 or
Mamu-A*01/SL8 tetramer and antibodies to ?4?7and ?E?7.
VOL. 77, 2003 CTLs REDUCE ACUTE-PHASE VIRAL REPLICATION 13355
Vaccinated macaques do not control pathogenic SIVmac239.
After mucosal challenge with SIVmac239, control animals ex-
perienced peak viremia at 2 weeks postchallenge of between 3
? 106and 1.2 ? 107viral RNA/ml of plasma, with a mean of
7.6 ? 106copies/ml (Fig. 9). In contrast, vaccinated animals
demonstrated significantly lower peak viral loads than the con-
trols, with a mean viral load of 4 ? 105viral copies/ml, ?1 log
less than the mean viral load of the controls (P ? 0.005). At 3
weeks postinfection and at various time points thereafter, the
difference in viral loads between the vaccinees and controls lost
statistical significance. This suggests that the vaccine-induced
CTL were able to reduce initial viral replication but were
unable to control replication once the infection became estab-
Neutralizing antibodies. Since Env was not included as an
immunogen in the vaccination regimen, we did not expect
neutralizing antibodies to be present at the time of challenge.
However, since vaccinees did initially control virus replication
relative to controls, we tested for the presence of neutralizing
antibodies 6 months after challenge. Little or no neutralizing
antibody activity against SIVmac239 was seen in the sera of
either vaccinated or control animals tested against a human
PBMC-grown stock of SIVmac239 and measured with
CEMx174 as the target cells. However, all of the animals did
show neutralizing antibody responses to a T-cell-line-adapted
strain of SIVmac251, as measured on CEMx174 cells (Table
We used a DNA prime/rMVA boost regimen to induce HTL
and a strong, multispecific CTL response. These CTL re-
sponses were detected in both systemic and mucosal tissues. By
delivering two rMVA boosts via different routes, we were able
to elicit the highest SIV epitope-specific CTL responses re-
ported thus far for the DNA/rMVA strategy. In vaccinees, an
average of 12% of their CD3?CD8?lymphocytes were
Mamu-A*01/CM9 tetramer-positive 1 week after an i.v. boost
with rMVA. These vaccine-induced Gag181-189CM9-specific
tetramer levels were up to 2-fold higher than those achieved in
a similar study using a DNA prime/rMVA boost strategy (6),
?10-fold higher than tetramer levels in a cytokine-augmented
DNA vaccination regimen (8), and approximately half of those
induced by a DNA/CRL1005 prime/adenovirus boost regimen
(50). Moreover, the i.v. boost induced long-lived mucosal CTL,
as shown by the expression of mucosal homing and retention
markers on PBMC and detection of Gag181-189CM9-specific
CTLs in the sigmoid colon for up to 10 weeks postboost.
Our vaccine strategy differed from those reported previously
in that we used the second i.v. rMVA boost to target CTL to
the mucosa. Targeting vaccine-induced immune responses to
mucosal surfaces may be important for several reasons. Mu-
cosal surfaces provide the first line of defense against sexually
transmitted HIV, and locating virus-specific CTL in the mu-
cosa may facilitate a rapid response after exposure. In a murine
study, virus-specific memory T cells derived from tertiary lym-
phoid tissues, including the intestinal mucosa, are more di-
rectly lytic than their splenic counterparts. Therefore, vaccine-
induced mucosal CTL may be more effective at limiting early
HIV/SIV replication. Since the vast majority of HIV infections
occur across mucosal barriers, it may be crucial for successful
HIV vaccines to elicit both systemic and mucosal responses.
Our vaccine regimen successfully elicited ?4?7
CTL and tetramer-positive CD8?cells in mucosal tissues.
After i.r. challenge with a high dose of SIVmac239, a strong
anamnestic CTL response significantly reduced peak viremia
in the vaccinated animals (P ? 0.005). After peak viremia, the
difference in the viral loads between the vaccinees and the
controls lost statistical significance and none of the vaccinated
FIG. 7. Tetramer-positive lymphocytes are still detectable in PBMC and IEL/LPL at 10 weeks after the rMVA boost (i.v.). Fresh PBMC (upper
panel) or IEL/LPL obtained from sigmoid colon punch biopsies (lower panel) at 10 weeks after the second rMVA boost (i.v.) were stained with
anti-CD3, anti-CD8, and Mamu-A*01/CM9 tetramer.
13356VOGEL ET AL. J. VIROL.
animals were able to control viral replication in the chronic
phase. Neutralizing antibodies to SIVmac239 were not de-
tected in any of the animals at 6 months postchallenge. It is
possible that with an increased number of animals in the study
a statistically significant difference in the viral loads may have
been maintained by the vaccinees into the chronic phase of
infection. However, even with a small sample size, the present
study suggests that multispecific CTLs are capable of providing
a degree of protection against acute viral replication but, with-
out neutralizing antibodies, they are unable to control chronic
viral replication after a high-dose mucosal challenge of patho-
Taken together, our data suggest that even very vigorous
CTL responses, targeted both systemically and mucosally, can-
not alone control a pathogenic SIV challenge. In contrast,
some recent reports have suggested that CTL-based vaccines
can ameliorate the course of immunodeficiency diseases (6–8,
50). These studies used the chimeric virus SHIV-89.6P as the
challenge, and the discrepancy between our results and those
of other groups can likely be attributed to fundamental differ-
ences between SHIV-89.6P and SIVmac239. First, SHIV-89.6P
and SIVmac239 exhibit different cell tropisms. HIV, SIV, and
SHIV can be phenotyped based on the coreceptor used for cell
attachment (10). The major coreceptors for HIV and SIV are
the chemokine receptors CCR5 and CXCR4 (11). SIVmac239
is a CCR5-utilizing (R5) virus, and infected macaques typically
show a gradual loss of peripheral CD4?T cells; this loss is
analogous to that seen in the course of most HIV infections
(18, 25, 28, 29, 31, 55). SHIV-89.6P, meanwhile, expresses an
env gene derived from a dualtropic virus that could use CCR5
or CXCR4 for entry (R5X4) (42, 55). Macaques infected with
SHIV-89.6P show a rapid and irreversible loss of CD4?T
lymphocytes in the peripheral lymphoid tissues that is similar
to that seen in infections with CXCR4-using (X4) strains of
HIV (17, 26, 30, 41–43, 47, 49). Moreover, SHIV-89.6P, unlike
most primary strains of HIV, is sensitive to neutralizing anti-
bodies (35, 41), whereas our data and those of others show that
SIVmac239 is difficult to neutralize (16, 23, 24). A previous
study linked the ability of macaques to mount an antibody
FIG. 8. Cellular immune response to Gag181-189CM9 and Tat28-35SL8 at 2 weeks postchallenge. Fresh PBMC was isolated at 2 weeks
postinfection and tested for IFN-? production upon peptide stimulation with Gag181-189CM9 and Tat28-35SL8. The frequency of CD8?tetramer-
positive lymphocytes (A) correlates with the proportion of CD8?lymphocytes producing IFN-? in response to the respective peptides (B), but at
a somewhat lower frequency.
VOL. 77, 2003 CTLs REDUCE ACUTE-PHASE VIRAL REPLICATION13357
response to SHIV-89.6P to longer survival (30), suggesting that
antibodies play a significant role in the observed protection
from disease progression. Thus, in recent vaccine studies with
SHIV-89.6P as the challenge virus (6–8, 45, 50), protection of
CD4?T cells from rapid depletion may have been the key to
the vaccinees’ long-term survival. If preserved, CD4?T cells
could provide adequate help to B cells, enabling them to
mount an effective antibody response against neutralization-
susceptible SHIV-89.6P. Furthermore, in at least three of these
previous vaccine studies the viral envelope was used as an
immunogen and, as a result, cross-reactive neutralizing anti-
bodies may have developed rapidly after challenge. It is diffi-
cult to understand the rationale for using SHIV-89.6P in vac-
cine studies designed to test the efficacy of CTL-based vaccines
when several SIV strains (such as SIVmac251, SIVmac239, and
FIG. 9. Viral loads for all animals. (A) The virus load over time was plotted for each animal. Blue traces, vaccinees; red traces, controls.
(B) Geometric mean virus loads for vaccine (blue) and control (red) groups. The viral loads were determined by kinetic PCR.
TABLE 4. Neutralizing antibodies from vaccinated and control
animals at 6 months postchallenge with SIVmac239
Nab titer toa:
aNab titer, the reciprocal serum dilution at which 50% of the cells were
protected from virus-induced killing, as measured by neutral red uptake.
bTCLA, T-cell line adapted.
13358VOGEL ET AL. J. VIROL.
SIVmacE660) are readily available. In contrast to SHIV-89.6P,
SIVmac239 resembles HIV in that it is very difficult to neu-
tralize with antibodies (23).
Despite strong CTL responses, including mucosally located
responses, vaccinated macaques lost control of SIVmac239 by
the chronic phase. This failure to control virus replication may
be the result of several factors. Both the vaccinees and the
controls were infected with a single, high dose of SIVmac239.
Although several factors play a role in HIV transmission, re-
cent studies have suggested that the viral load of the infected
individual is the chief predictor of heterosexual transmission
(20, 39). These studies show that the probability of transmis-
sion increases with rising virus levels. In addition, an empirical
model of heterosexual HIV-1 transmission predicts that, when
seminal levels in plasma are high (?100,000 copies/ml), trans-
mission occurs in 1 of 100 sexual encounters, whereas the
probability of transmission declines rapidly with decreasing
seminal viral loads (15). Thus, it may be that the single dose of
SIVmac239 used to infect macaques is unnaturally high and
does not accurately reflect the transmission of HIV in humans.
In a challenge more closely imitating physiological conditions,
vaccine-induced CTL, such as those we observed, may be able
to control viral replication. However, the development of virus-
specific CTL alone may not be sufficient to limit viral replica-
tion. Additional help from neutralizing antibodies and the in-
nate immune system may be necessary to control HIV
replication. Moreover, the induction of particularly strong im-
munodominant CTL responses, like those against Gag181-
189CM9 and Tat28-35SL8, may hinder the stimulation of sub-
dominant CTL responses during infection. The lack of
activation of subdominant CTL responses may impede the
immune system’s ability to mount an effective response.
In conclusion, the DNA prime/rMVA boost vaccination reg-
imen generated HTL and long-lived, multispecific systemic and
mucosal CTL. After mucosal challenge with the highly patho-
genic SIVmac239, a massive anamnestic CTL response was
observed in the limited number of vaccinees, and these animals
controlled peak viral replication (P ? 0.005). In contrast to
similar studies with SHIV-89.6P as the challenge virus, our
vaccinated animals were unable to control viral replication in
the chronic phase of infection. The present study suggests that
multispecific CTL, in the absence of neutralizing antibodies,
can achieve a modicum of control over early viral replication
but are unable to control chronic viral replication after a high
dose mucosal challenge with a pathogenic SIV.
T.U.V. and M.R.R. contributed equally to this study.
We thank Ronald Desrosiers for providing the SIVmac239, Mari-
anne Lo ¨wel for expertise in preparing the MVA, Sarah Fuenger for
help with ICS, Kim Schmidt for technical assistance, and Thomas
Friedrich for help in preparation of the manuscript. The anti-?4?7
antibody was generously provided by Millennium Pharmaceuticals.
This work is supported by NIH grants AI41913, AI46366, AI45461,
RR15371, and RR00169 and by grants from the Deutsche Forschungs-
gemeinschaft and the European Community (QLK2-2000-1040 to
G.S.). D.I.W. is a recipient of an Elizabeth Glaser scientist award.
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