JOURNAL OF VIROLOGY, Dec. 2011, p. 12708–12720
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 23
Vaccine Protection against Simian Immunodeficiency Virus in
Monkeys Using Recombinant Gamma-2 Herpesvirus?
John P. Bilello,1† Julieta M. Manrique,1‡ Young C. Shin,1William Lauer,1Wenjun Li,2
Jeffrey D. Lifson,3Keith G. Mansfield,1R. Paul Johnson,1and Ronald C. Desrosiers1*
New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-91021; University of
Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 016552; and AIDS and
Cancer Virus Program, SAIC Frederick Inc., National Cancer Institute, NCI Frederick,
Frederick, Maryland 217023
Received 28 April 2011/Accepted 27 August 2011
Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in
which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus
(SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each
recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive
rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of
SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive
anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC)
tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much
greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two
monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization,
responses measured by MHC tetramer staining in the two Mamu-A*01?RRV-negative monkeys reached 9.3%
and 13.1% of all CD8?T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8?T
cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8?T cell responses persisted at high levels
up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory
phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and
despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody
responses were below our ability to detect them. Immunized monkeys, together with three unimmunized
controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized
monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log10units lower
and chronic-phase viral loads were 1.0 to 3.0 log10units lower in immunized animals than the geometric mean
of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses follow-
ing challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate
the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live,
replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated
strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be
The difficulties in finding an effective vaccine against human
immunodeficiency virus type 1 (HIV-1)/AIDS revolve princi-
pally around the properties of HIV-1 itself. Once HIV-1 es-
tablishes an initial infection, it has an uncanny ability to rep-
licate persistently and relentlessly despite apparently strong
virus-specific humoral and cellular immune responses. It is
now clear that HIV, as well as its monkey counterpart simian
immunodeficiency virus (SIV), has evolved specific strategies
to evade intrinsic, innate, and adaptive immunity (16, 33). For
adaptive immunity, the evasion strategies are multifaceted.
These retroviruses have error-prone reverse transcriptases to
replicate their genetic information and are able to quickly
select for immune escape variants (both humoral and cellular)
that are able to replicate efficiently in the face of ongoing
immune responses (8, 9, 24, 53). The viruses have constructed
their envelope glycoprotein spikes in a way that makes it dif-
ficult for antibodies to access and difficult for them to neutral-
ize viral infectivity (10, 22, 49, 57). One of the viral gene
products, Nef, downregulates major histocompatibility com-
plex (MHC) class I molecules from the surfaces of infected
cells, making these infected cells less susceptible targets of
virus-specific CD8?T cells (13, 58, 60). There is enormous
sequence variability in HIV-1 circulating in the population,
and it is not clear how a vaccine will be able to provide pro-
tection against this great diversity of sequences. Finally, the
main target cell for viral replication is the CD4?T lymphocyte,
a major orchestrator of effective immune responses.
One vaccine approach that has worked reasonably well—
much better than other vaccine approaches, in fact—against
* Corresponding author. Mailing address: New England Primate
Research Center, One Pine Hill Drive, Box 9102, Southborough, MA
01772-9102. Phone: (508) 624-8042. Fax: (508) 624-8190. E-mail:
† Present address: Idenix Pharmaceuticals, Inc., 60 Hampshire
Street, Cambridge, MA 02139.
‡ Present address: Estacio ´n de Fotobiologia Playa Unio ´n, Casilla de
Correo 15, (9103) Rawson, Chubut, Argentina.
?Published ahead of print on 7 September 2011.
SIV in monkeys is live attenuated deletion mutant strains of
virus (14, 31, 68). While live attenuated strains of SIV with Nef
deleted have protected well against homologous challenge or
challenge by strains closely matched in sequence, even live
attenuated SIV has not performed so well against challenge by
heterologous strains not closely matched in sequence (52, 67).
This inability to protect well against sequence-mismatched
strains of SIV may be analogous to the inability of infection by
one HIV-1 strain to routinely protect against superinfection by
a different circulating strain of HIV-1 (5, 50).
Through complex genetic engineering, strains of SIV that
are capable of a single round of infection and a single round of
virus production but are incapable of further spread to other
cells have been constructed (19, 20). Repeated administrations
of such “single-cycle SIV” can provide some level of protection
against subsequent challenge by homologous SIV, but the de-
gree of protection is nowhere near as great as that observed
with live attenuated strains (30). One possible explanation for
this observation is that anamnestic immune responses may
simply not be sufficient to respond in time to blunt replication
of a wild-type challenge strain, even one that is totally matched
Herpesviruses provide one potential means to examine
the importance of persistent antigen expression for the de-
gree of protection. Herpesviruses have large double-
stranded DNA genomes that can stably accommodate large
amounts of inserted foreign DNA. They persist for life, and
immune responses to them in terms of effector cells persist
at readily detectable levels for life (59). Eight distinct hu-
man herpesviruses have been identified, and each falls into
one of the distinct subgroups: alphaherpesvirus, betaherpes-
virus, gamma-1 herpesvirus, and gamma-2 herpesvirus. Her-
pesviruses from different subgroups can be quite distinctly
different, targeting different types of cells for primary rep-
lication and different types of cells for persistence, and they
even carry different compositions of genes. To date, the use
of recombinant herpesvirus vectors to express SIV antigens
in monkeys has been reported for the alphaherpesvirus her-
pes simplex virus (HSV) (35, 47) and for the betaherpesvirus
cytomegalovirus (CMV) (26).
Here, we report the construction and performance of re-
combinants of the gamma-2 herpesvirus of rhesus monkeys,
rhesus monkey rhadinovirus (RRV) (2, 17).
MATERIALS AND METHODS
Cell culture. Human embryonic kidney cells (293T) were maintained on Dul-
becco modified Eagle medium (DMEM) supplemented with 10% fetal calf
serum, 2 mM L-glutamine, and penicillin-streptomycin (50 IU and 50 ?g/ml,
respectively) at 37°C in a humidified incubator with 5% CO2. Rhesus macaque
skin fibroblasts (RF) were maintained in DH20 (DMEM supplemented with
20% fetal calf serum, 2 mM L-glutamine, penicillin-streptomycin [50 IU and 50
?g/ml, respectively], and 10 mM HEPES) at 37°C in a humidified incubator with
Cosmid cloning. Procedures for insertion of foreign sequences upstream of the
RRV R1 reading frame in the leftmost cosmid clone, ah28?A/H, have been
described previously (6). Specific features of the recombinants used for the
current study are illustrated in Fig. 1 and described here. For insertion of the
expression-optimized SIV envelope gene driven by the elongation factor 1 pro-
moter (EF1-SIVenv-eo), complementary oligonucleotides, 5?-CTAGTTGTTTA
AACGGGGCGCCGGA-3? and 5?-CTAGTCCGGCGCCCCGTTTAAACAA-
3?, were annealed at 55°C and phosphorylated using T4 polynucleotide kinase,
forming an SpeI-PmeI-SpeI adaptomer. The adaptomer featured a cut SpeI site
at each end flanking a central PmeI site. For insertion of the codon-optimized
SIV gag gene driven by the CMV promoter (CMV-SIVgag) and the SIV rev-tat-
nef fusion construct driven by a simian virus 40 (SV40) promoter (SV40-
SIVRTN), complementary oligonucleotides, 5?-CTAGTGGCTAGGGATAAC
AGGGTAATA-3? and 5?-CTAGTATTACCCTGTTATCCCTAGCCA-3?, were
annealed and phosphorylated as before to form an SpeI-ISceI-SpeI adaptomer.
The adaptomer featured a cut SpeI site at each end flanking a central ISceI site.
The ah28?A/H cosmid was linearized at base pair 206 with SpeI and dephos-
phorylated using calf intestinal phosphatase (CIP). Subsequently, the linearized
ah28?A/H cosmid was ligated to the SpeI-PmeI-SpeI or SpeI-ISceI-SpeI adap-
tomer, yielding ah28?A/H-PmeI or ah28?A/H-ISceI, respectively.
Each SIV expression insert was designed to be noncomplementary to the
others in order to avoid recombination events when subsequent SIV-recombi-
nant RRV viruses were used to coinfect monkeys. To generate the ah28?A/H
EF1-SIVenv cosmid (Fig. 1), expression-optimized SIVenv sequences were ex-
cised from a modified p64s S23T plasmid (obtained from E. Yuste, New England
Primate Research Center [NEPRC], Southborough, MA) and ligated into pEF1
p(A), a pEF1-mycHisA plasmid (Invitrogen) that was altered to contain (i) an
HSV thymidine kinase poly(A) sequence, HSVtk p(A), downstream from the
XbaI site within the plasmid and (ii) an additional PmeI restriction endonuclease
site upstream from the EF1 promoter. Briefly, the pEF1-mycHisA plasmid was
digested with NotI and XbaI and ligated to an adaptomer containing the HSVtk
p(A) sequence flanked by NotI and XbaI. This adaptomer was formed in the
same manner described above using complementary oligonucleotides, 5?-GGC
CGCAATAAAAAGACAGAATAAAT-3? and 5?-CTAGATTTATTCTGTCTT
TTTATTGC-3?. To insert the PmeI restriction endonuclease site upstream from
the EF1 promoter, an adaptomer containing the PmeI restriction site flanked by
MluI restriction sites was formed in the same manner as described above using
complementary oligonucleotides, 5?-CGCGTTGTTTAAACGGGGCGCCGG
A-3? and 5?-CGCGTCCGGCGCCCCGTTTAAACAA-3?. The pEF1-mycHisA
plasmid was digested with MluI and ligated to this adaptomer. The p64s S23T
plasmid was modified to contain a KpnI restriction endonuclease recognition site
by the ligation of a EcoRI-KpnI-EcoRI adaptomer into the EcoRI site just
upstream from the expression-optimized SIVenv gene. This adaptomer was
formed in the same manner as described above using complementary oligonu-
cleotides, 5?-AATTCCGCGGATCCGCGGGGTACCG-3? and 5?-AATTCGGT
ACCCCGCGGATCCGCGG-3?. Finally, pEF1 p(A) and the modified p64s
S23T were digested with KpnI and gel extracted. Following dephosphorylation of
pEF1 p(A) with CIP (NEB), the two products were ligated together to make the
pEF1-64s plasmid. The ah28?A/H-PmeI cosmid was digested with PmeI, de-
phosphorylated with CIP, and gel extracted using the QiaExII kit (Qiagen). The
expression-optimized SIV env gene driven by the EF1 promoter was excised from
the pEF1-64s plasmid by digestion with PmeI, gel extracted, and ligated to the
ah28?A/H-PmeI fragment to generate the ah28?A/H EF1-SIVenv cosmid.
To generate the ah28?A/H SV40-RTN cosmid (Fig. 1), the SIV rev-tat-nef
(RTN) sequence was excised from the pcDNA/RTN plasmid (the kind gift of
David Knipe, Harvard Medical School) by digestion with BamHI and ligated into
a modified pSG5 plasmid that was digested with BamHI and dephosphorylated
using CIP. The pSG5 plasmid (Stratagene) was modified to contain the SV40
promoter, a multicloning site containing a single BamHI restriction endonu-
clease site, and the SV40 poly(A) sequence flanked by ISceI restriction endo-
nuclease recognition sites, giving rise to the pSG5-RTN-B plasmid. The ISceI
site upstream from the SIV-RTN sequence was generated by QuikChange (Agi-
lent Technologies) mutagenesis following the manufacturer’s protocol using the
following oligonucleotides: 5?-CGGCCAGTGAATTGTCGACTAGTGAGGC
GGAAAGAACCAGCTG-3? and 5?-CAGCTGGTTCTTTCCGCCTCACTAG
TCGACAATTCACTGGCCG-3?. The ISceI site downstream from SIV-RTN
was created by insertion of a BglII-ISceI-BglII adaptomer formed as described
above using complementary oligonucleotides, 5?-GATCTGGCTAGGGATAAC
AGGGTAATA-3? and 5?-GATCTATTACCCTGTTATCCCTAGCCA-3?. The
ah28?A/H-ISceI cosmid was digested with ISceI, dephosphorylated with CIP,
and gel extracted using the QiaExII kit (Qiagen). The SIV-RTN sequence driven
by the SV40 promoter was excised from the modified pSG5 plasmid by digestion
with ISceI, gel extracted, and ligated to the ah28?A/H-ISceI fragment to gen-
erate the ah28?A/H SV40-RTN cosmid. Insertion of the SV40 SIV-RTN frag-
ment occurred in the antisense orientation of the ah28?A/H ISceI cosmid rel-
ative to the R1 reading frame in this cosmid.
To generate the ah28?A/H CMV-SIVgag cosmid (Fig. 1), the codon-opti-
mized SIV gag sequence was excised from the pTkdGag plasmid and inserted
into a modified pcDNA3.1? (Invitrogen, Carlsbad, CA) plasmid containing the
CMV immediate-early promoter (CMV-IE) and the bovine growth hormone
(BGH) poly(A) sequence. An ISceI restriction endonuclease sequence was in-
troduced both 5? and 3? of the CMV-IE promoter-SIV gag-BGH poly(A) se-
VOL. 85, 2011VACCINE PROTECTION AGAINST SIV IN MONKEYS12709
quence in a manner similar to that described above, giving rise to the pcDNA/gag
plasmid. The codon-optimized SIV-gag sequence driven by the CMV promoter
was excised from pcDNA/gag by digestion with ISceI, gel extracted, and ligated
to the ah28?A/H-ISceI fragment to generate the ah28?A/H CMV-SIVgag
DNA sequencing. Cosmid and plasmid constructs were sequenced with a CEQ
8000 Genetic Analysis System using a dye terminator cycle-sequencing kit as
specified by the manufacturer (Beckman Coulter, Fullerton, CA).
Cotransfection and virus preparation. Prior to transfection, overlapping cos-
mids necessary to reconstruct the RRV26-95 genome, including either the
FIG. 2. Demonstration of expression of the desired SIV proteins in rhesus fibroblast cultures infected with RRV-SIV recombinants. Cell lysates
were prepared, proteins were separated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were Western blotted to a filter
and reacted with SIV-specific antibodies. (A) RRV-env1 and RRV-env2 represent lysates from cells infected with two independently derived stocks
of RRV-SIVenv. RRV26-95 and RRV-GFP are also infected-cell lysates. pEF1-64s is a cell lysate from 293T cells transfected with the expression
plasmid for SIVmac239 Env. In the top gel, blots were reacted with an antibody specific for the Env surface subunit (gp120), and in the lower gel,
blots were reacted with a monoclonal antibody that recognizes the Env transmembrane subunit (gp41). (B) RRV-gagA and RRV-gagB represent
lysates from cells infected with two independently derived stocks of RRV-SIVgag. pcDNA/gag is a cell lysate from 293T cells transfected with the
expression plasmid for codon-optimized SIV gag. (C) Western blots for detection of Rev-Tat-Nef fusion protein. RRV-RTN-2, -3, -5, and -6
represent lysates from cells infected with four independently derived stocks of RRV-SIVrev-tat-nef. SG5-RTN-B is a cell lysate from 293T cells
transfected with the expression plasmid for the Rev-Tat-Nef fusion protein.
FIG. 1. Schematic representation of recombinant RRV-SIV constructions. The site of insertion into the leftmost RRV cosmid clone is as
described by Bilello et al. (6). The transcriptional elongation factor 1 promoter region was used to drive expression of a codon-optimized
SIVmac239 gp160 envelope sequence. The CMV immediate-early promoter was used to drive expression of a codon-optimized SIVmac239 Gag
sequence, and the SV40 promoter was used to drive expression of a Rev-Tat-Nef fusion protein.
12710 BILELLO ET AL.J. VIROL.
ah28?A/H EF1-SIVenv, ah28?A/H CMV-SIVgag, or ah28?A/H SV40-RTN
cosmid, were digested overnight with the ICeuI homing endonuclease to remove
the RRV26-95 sequence from the pSuperCos 1 backbone vector as described by
Bilello et al. (6). The cosmid DNA was precipitated by adding 3 volumes of 5%
3 M sodium acetate/95% ethanol and incubating it for ?1 h at ?20°C. The DNA
was then pelleted by centrifugation for 10 min at maximum speed in a micro-
centrifuge. The pellets were washed in 70% ethanol, dried, and rehydrated in
H2O. One day postseeding, 293T cells (4.5 ? 105cells/well in 6-well plates) were
transfected with different combinations of digested overlapping cosmids (0.4 ?g
each cosmid) using Transfectin reagent (Bio-Rad Laboratories, Hercules, CA)
following a scaled-down procedure. As a positive control, 0.25 ?g of whole viral
RRV DNA isolated from column-purified RRV26-95 was transfected in the
same manner. At 5 days posttransfection, cell-free culture supernatant was col-
lected and stored at 4°C. To amplify recombinant stocks generated in 293T cells,
fresh rhesus monkey fibroblast cultures were inoculated with 1 ml of the super-
natant collected from the 293T transfections. The inoculated fibroblast cultures
were passaged until the emergence of viral plaques was observed in the cultures,
and then the cultures were maintained without splitting until complete lysis of
the fibroblast monolayer occurred. High-titer recombinant RRV stocks were
subsequently generated in fresh rhesus fibroblast cultures.
Isolation and analysis of RRV DNA. For each RRV, supernatant collected
following complete lysis of RRV-infected rhesus fibroblasts was subjected to
low-speed centrifugation to remove cellular debris. The supernatant was then
filtered through a 0.45-?m-pore-size filter to remove any additional debris. The
filtered supernatant was then centrifuged for 3 h at 45,000 ? g in a Sorvall Type
19 rotor to pellet the virus. The crude virus was resuspended in Tris-EDTA (TE)
buffer and lysed by adding 0.1 volume 1% N-lauroylsarcosine and proteinase K
and incubating it at 60°C for 1 h. The mixture was extracted twice with phenol-
chloroform, followed by four chloroform washes. The DNA was recovered by
precipitation with 2.5 volumes 5% 3 M sodium acetate/95% ethanol, rinsed in
80% ethanol, and resuspended in Tris-EDTA buffer. Viral DNA was digested
with restriction endonucleases, separated on a 0.5% agarose electrophoretic gel,
and stained with ethidium bromide.
Plaque assay. The titers of parental RRV26-95 and recombinant RRV stocks
were determined as previously described (18). Briefly, cell-free culture superna-
tant was collected following complete lysis of RRV-infected rhesus fibroblasts.
FIG. 3. Restriction endonuclease analysis of recombinant RRV-SIV strains confirms the expected genetic changes. DNA was extracted from
concentrated virions and digested with the indicated restriction endonucleases, and the fragmented DNA was analyzed by agarose gel electro-
phoresis. U, undigested; E, EcoRI; B, BamHI. Fragments with altered mobility due to the insertion of an SIV expression cassette are marked with
TABLE 1. Characteristics of rhesus monkeys used in vaccine trials
Mamu A*01 Mamu A*02Mamu B*17 Mamu B*08
aDOV, day of vaccination. 440-92 and 247-04 were already RRV positive as a consequence of natural RRV infection at the time of RRV-SIV administration.
bDOC, day of challenge.
cMonkey was naturally RRV positive at the time of enrollment.
d?, positive; ?, negative.
VOL. 85, 2011VACCINE PROTECTION AGAINST SIV IN MONKEYS 12711
Fresh fibroblasts were seeded into 12-well plates at 2 ? 105cells/well. The
following day, 10-fold serial dilutions of the virus-containing supernatant were
made in DH20. The media were removed from the RF cultures and replaced
with 200 ?l of diluted virus/well. The cultures were then incubated for 1 h at 37°C
with gentle rocking every 15 min. After 1 h, 2 ml of Hank’s buffered saline
solution (HBSS) was added to each well and subsequently aspirated. Two mil-
liliters of overlay medium (a 1:1 ratio of 2? DMEM and 1.5% methylcellulose
[Sigma, St. Louis, MO] supplemented with 2% fetal calf serum) was applied, and
the cultures were incubated at 37°C and 5% CO2for 1 week. The overlay
medium was aspirated, and a staining solution (0.8% crystal violet in 50%
ethanol) was applied for 10 min. Each well was washed 5 times with distilled
water, and the number of plaques at each dilution of inoculum was determined.
Transfection of positive-control plasmids. One day postseeding, 293T cells
(4.5 ? 105cells/well in 6-well plates) were transfected with either the pEF1-64s
(for SIV env), pcDNA/gag, or SG5-RTN-B (for SIV rev-tat-nef) expression plas-
mid using Transfectin reagent (Bio-Rad Laboratories, Hercules, CA) following a
Immunoblotting. At 48 h posttransfection or 4 days postinfection, cultures
were rinsed with phosphate-buffered saline (PBS) and lysed with RIPA buffer
(Boston BioProducts, Boston, MA). The lysates were sonicated at 20% for 10 s,
and the debris was spun down at maximum speed in a microcentrifuge for 1 min.
These proteins or those obtained by lysis of SIVmac239 isolates were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene difluoride (PVDF) membranes. The membranes
were blocked overnight at room temperature in 5% milk in phosphate-buffered
saline containing 0.1% Tween 20 (PBS-T; Sigma, St. Louis, MO). Primary anti-
body to SIVenv gp41/gp160 (KK41 from the NIH AIDS reagent repository),
SIVenv gp120 (3.11H; a kind gift from James Robinson, Tulane University),
SIVgag (N27), or SIV-nef (from the NIH AIDS reagent repository) diluted in
5% milk in PBS-T was applied and rocked at room temperature for 1 h. After
successive washings in PBS-T, the blots were incubated in secondary anti-mouse
(Santa Cruz) diluted in 5% milk in PBS-T. The blots were washed in PBS-T, and
antibody binding was detected using the SuperSignal West Pico chemilumines-
cent reagent (Pierce) and a Fuji LAS3000 chemiluminescence imager (Bio-Rad).
Antibody detection by ELISA. Purification of RRV, coating of enzyme-linked
immunosorbent assay (ELISA) plates, and detection of anti-RRV antibody re-
sponses by ELISA have been described previously (45). Detection of anti-SIV
antibody responses by ELISA used procedures described by Desrosiers et al. (15)
except that SIVmac239 gp120 protein was used for the coating of ELISA plates.
IFN-? ELISPOT assays. SIV-specific T cell responses were enumerated using
an enzyme-linked immunospot (ELISPOT) assay for detection of macaque
gamma interferon (IFN-?) (Mabtech, Mariemont, OH) using standard method-
ologies as previously described (1, 63). Briefly, freshly isolated peripheral blood
mononuclear cells (PBMC) in R10 medium were plated in duplicate at 3 ? 105
cells/well and incubated with overlapping peptide pools corresponding to the
SIVmac239 Gag, Env, Rev, Tat, and Nef sequences at a final concentration of 2
?g/ml for individual peptides. The numbers of spots (representing IFN-?-pro-
ducing T cells) were detected in a colorimetric assay for bound IFN-? and were
counted using an automated ELISPOT plate reader (Zellnet Consulting, New
York, NY). To determine the absolute numbers of SIV-specific IFN-?-producing
T cells per 106PBMC, the number of background spots in medium-only control
wells was subtracted from the number of spots in peptide-stimulated wells.
Concanavalin A-stimulated PBMC served as a positive control. The peptides
were obtained from the AIDS Research and Reference Reagent Program, Di-
vision of AIDS, NIAID, NIH, or were synthesized by the Massachusetts General
Hospital Peptide Core Facility, Boston, MA.
Viral load measurement. Plasma viral loads were determined by real-time
PCR methods as described previously (12). For each sample, 1.5 ml of plasma
was processed to yield a threshold limit of detection of 10 Gag RNA copy
equivalents per ml.
Flow cytometric analysis. Staining and phenotyping of SIV tetramer binding
cells was performed as previously described (56, 61) with modifications as noted
below. Briefly, 200 ?l of whole blood was incubated with titrated amounts of
tetramers in 100 ?l PBS with 2% fetal bovine serum (FBS) at 37°C and 5% CO2
for 60 min in the dark and then stained with the monoclonal antibodies indicated
FIG. 4. Detection of SIVgag in peripheral lymph node biopsy specimens by immunohistochemistry. (A) SIV Gag-expressing cells in a lymph
node biopsy specimen from animal 175-91 (inset: positive cell) inguinal lymph node; DAB chromogen. (B) Irrelevant antibody control.
TABLE 2. Localization of Gag-expressing
cells by immunohistochemistry
a?, small, and ??, moderate numbers of Gag-positive cells. IHC, immuno-
12712 BILELLO ET AL.J. VIROL.
below. For demonstration of the effector memory phenotype, PBMC (1 ? 106to
2 ? 106) were incubated with tetramers for 45 min in the dark prior to staining
with monoclonal antibodies. Mamu-A*01 class I tetramers conjugated to phy-
coerythrin (PE) and complexed with the A*01 Gag181-189CM9 (4) and A*01
Tat28-35SL8 (3, 38) epitopes were kindly provided by Nancy Wilson and David
Watkins (Wisconsin National Primate Research Center, Madison WI). For in-
tracellular staining, samples were fixed with Caltag Fix and Perm Solution A
(Burlingame, CA) for 10 min at room temperature, washed, incubated with
intracellular antibodies in Caltag Fix and Perm Solution B, and then processed
as described above. Samples were analyzed on a Becton Dickinson (BD)
FACSCalibur or BD LSR-II flow cytometer (BD Immunocytometry Systems,
San Jose, CA). Anti-mouse IgG(?/?)-negative polystyrene beads (BD Comp-
Beads; BD, CA) or PBMC stained separately with individual monoclonal anti-
bodies used in the test samples were used for electronic compensation. Gates for
the expression of perforin, CCR7, and CD127 were established using fluores-
cence-minus-one (FMO) controls (54).
The following fluorescently labeled monoclonal antibodies were obtained from
BD Biosciences (La Jolla, CA): CD3-fluorescein isothiocyanate (FITC) (SP34-
2), CD3-allophycocyanin (APC)-Cy7 (SP34-2), CD4-peridinin chlorophyll pro-
tein complex (PerCP) (L-200), CD8a-PerCP, CD8a-Alexa 700 (RPA-T8), and
CD28-PE-Texas Red (L293). Perforin-FITC (Pf-344) was purchased from
Mabtech (Cincinnati, OH). CCR7 (150503; R&D Systems, Minneapolis, MN)
was used as a custom Pacific Blue conjugate prepared at the NEPRC. All
antibodies were tested for cross-reactivity and titrated on rhesus macaque PBMC
to determine optimal staining concentrations and conditions.
Immunohistochemistry. Immunohistochemistry was performed on lymph
node biopsy specimens obtained 3 and 9 weeks following vaccination to identify
Gag-expressing cells using an avidin-biotin complex immunostain technique, as
previously described (28). Briefly, immunohistochemistry was performed on for-
malin-fixed paraffin-embedded tissue sections, which were deparaffinized in xy-
lene and rehydrated through graded ethanol to distilled water. Incubation in 3%
H2O2was used to block endogenous peroxidase activity and was followed by 60
min of incubation with the primary monoclonal antibody directed against SIVp27
(clone 183-H12-5C; NIH AIDS Research and Reference Reagent Program). The
sections were sequentially treated with a biotinylated secondary antibody and
horseradish peroxidase-conjugated streptavidin. The chromogenic substrate 3,3?-
diaminobenzidine (DAB) (Dako Corp.) was used to localize antigen-antibody
complexes. Tissue sections were counterstained with Mayer’s hematoxylin
(Sigma-Aldrich, St. Louis, MO), cleared, and coverslipped with permanent
mounting medium. An isotype-matched irrelevant control antibody was used on
all sections to control for nonspecific staining.
Animal studies. Rhesus macaques (Macaca mulatta) of Indian ancestry were
specific pathogen free of B virus, simian T-lymphotropic virus, simian retrovirus,
and SIV. The animals were housed at the NEPRC and maintained in accordance
with the Guide for the Care and Use of Laboratory Animals of the Institute of
Laboratory Resources, National Research Council. The facility is accredited by
the Association for the Assessment and Accreditation of Laboratory Animal
Care International, and all work was approved by Harvard Medical School’s
Standing Committee on Animals. The RRV serostatus was evaluated by ELISA
prior to the initiation of the study as previously described (17). In the vaccine
phase of the study, five animals were intravenously inoculated with a mixture of
RRV-SIV recombinants (1 ? 105PFU of each recombinant), and one control
animal was inoculated with 3 ? 105PFU of the parental cloned RRV26-95. The
animals were followed prospectively with sequential blood draws and lymph node
biopsies performed at 3 and 9 weeks. At 18 weeks, these animals and two
additional seropositive controls were challenged intravenously (i.v.) with 10 in-
fectious doses of SIVmac239. Euthanasia criteria were developed prior to the
initiation of the study and were carried out in accordance with the recommen-
dations of the Panel on Euthanasia of the American Veterinary Medical Asso-
Statistical analyses. Geometric means and associated 95% confidence inter-
vals (CI) of peak viral loads were computed for vaccinated and control groups.
Between-group differences in log10-transformed peak viral loads were tested
using a two-sided t test. Between-group differences in geometric means were also
expressed as a ratio of the vaccinated group to the control group, as well as
percent reduction. Geometric means of set point viral loads (weeks 6 to 32) for
the vaccinated and control groups were estimated using a linear mixed model
with a random intercept for each subject. The data were centered at week 16
postchallenge (the midpoint between weeks 6 and 32). The model accounts for
the correlations among the repeated measurements on the same experimental
subject. Based on the same model, differences in set point values were estimated
and tested for statistical significance using the Wald test. The estimated differ-
ence was then expressed as a ratio and as percent reduction.
FIG. 5. SIV-specific IFN-? ELISPOT responses in RRV-SIV-vac-
cinated macaques (Mm). PBMC were obtained at the indicated time
points, and SIV-specific responses to the indicated SIV peptide pool
were calculated after subtraction of spots obtained in wells incubated
with R10 medium alone. (A) Vaccine phase. (B) Challenge phase.
(C) Challenge phase for the unvaccinated control monkeys. ?, mon-
keys that were already RRV positive at the time of RRV-SIV
VOL. 85, 2011 VACCINE PROTECTION AGAINST SIV IN MONKEYS12713
Construction of RRV recombinants. A genetic system that
employs overlapping cosmid clones of RRV has been de-
scribed (6). A unique ISceI site in the leftmost cosmid clone
(ah28) was used as the site of insertion of SIV gene expression
cassettes (6) (Fig. 1). The promoter region of cellular tran-
scriptional elongation factor 1 (EF1) was used to drive expres-
sion of a codon-optimized (expression-optimized) version of
the env gene of SIVmac239 (27, 51). The CMV immediate-
early promoter was used to drive expression of an expression-
optimized version of the SIVmac239 gag gene (27, 51), and the
SV40 promoter was used to drive expression of a rev-tat-nef
fusion construct (64). Replication-competent recombinant
RRV strains were generated by transfection of overlapping
cosmid clones and expanded on early-passage rhesus monkey
fibroblast cultures as described previously (6).
Permissive rhesus fibroblasts were infected in culture with
each recombinant RRV, and cell lysates were prepared and
analyzed for the presence of the appropriate SIV gene prod-
uct by immunoblotting using appropriate monoclonal anti-
bodies to individual products (Fig. 2). The recombinant
RRV-SIVenv made the expected SIV Env products gp160,
gp120, and gp41. These Env proteins were readily detected
and were as abundant as in 293T cells transfected with the
expression plasmid containing the codon-optimized se-
quences (Fig. 2A). The RRV-SIVgag recombinant ex-
pressed a Gag protein of the expected 55 kDa as the prin-
cipal product, as well as apparent proteolytic breakdown
products (Fig. 2B). The RRV-SIVrev-tat-nef recombinant
expressed a protein product of approximately 60 kDa, the
predicted mass of the Rev-Tat-Nef fusion protein, as well as
lower-molecular-weight (MW) bands that are likely proteo-
lytic breakdown products (Fig. 2C). The predicted mass of
the Rev-Tat-Nef fusion protein is 56 kDa.
RRV virions were purified, DNA was isolated, and the vi-
rion DNA was subjected to restriction endonuclease analysis
(Fig. 3). The restriction endonuclease BamHI was particularly
useful for discriminating left-end fragments in recombinant
versus parental DNA. In each case, a left-end BamHI fragment
present in the parental strain was lost and replaced in the
recombinant strains by a higher-MW band of the expected size
for the newly inserted fragment (Fig. 3). Digestion of RRV-
SIV recombinant virus DNA with SphI or SpeI restriction
endonuclease also showed changes in fragmentation patterns
indicative of the inserted SIV expression cassettes. These re-
striction endonuclease analyses revealed no additional changes
FIG. 6. Frequency of Mamu-A*01 Gag CM9 tetramer binding cells in macaque 166-91. PBMC were obtained at the indicated times
postinoculation (PI) from monkey 166-91 (RRV naive). The frequencies of tetramer binding cells in CD3?CD8?lymphocytes are shown. DOI,
day of inoculation.
12714BILELLO ET AL. J. VIROL.
to the composition of the virion DNA other than the recom-
binant DNA fragment that was introduced.
Vaccine phase. Five rhesus monkeys were enrolled in a vac-
cine study for evaluation of these RRV-SIV recombinants.
Three of the monkeys were RRV negative at the time of
enrollment, and two were intentionally selected as naturally
infected and RRV positive (Table 1). The use of two RRV-
positive monkeys allowed us to subsequently evaluate the im-
pact of prior RRV infection on the take of the recombinant
RRV vaccine strains. Four of the five monkeys were Mamu-
A*01 positive to allow convenient use of MHC tetramers for
the evaluation of virus-specific CD8?T cell responses to de-
fined epitopes (Table 1). A mixture of the three RRV-SIV
recombinant strains was made to contain equivalent numbers
of PFU, and the mixture was inoculated intravenously into
each of the five test animals on the day of vaccination (DOV)
(Table 1). One of the three control monkeys (309-04) was
experimentally infected with the parental cloned RRV26-95 at
this time, and the other two control monkeys (390-93 and
328-04) were naturally RRV positive (Table 1).
Immunohistochemistry was used to localize Gag-expressing
cells in peripheral lymph node biopsy specimens obtained from
vaccinated animals. Small to moderate numbers of Gag-ex-
pressing cells were identified (Table 2 and Fig. 4) in RRV-
seronegative animals receiving the RRV-SIV vaccine at 3
weeks postinoculation. Rare Gag-positive cells were also ob-
served in one of the two RRV-positive monkeys (440-92) that
received RRV-SIV. Cytoplasmic staining was observed primar-
ily in cells in the parafollicular cortex. No staining was ob-
served in animals receiving the cloned or uncloned RRV or in
irrelevant antibody control sections. Staining was absent in
biopsy specimens taken 9 weeks following inoculation.
SIV-specific T cell responses after inoculation with RRV-
SIV recombinants were followed using IFN-? ELISPOT assays
and MHC class I tetramers. IFN-? ELISPOT assays were car-
ried out using overlapping peptide pools corresponding to the
Gag, Env, Rev, Tat, and Nef open reading frames. In the
RRV-naive animals 166-91, 175-91, and 128-04, robust
ELISPOT responses to Gag were observed 3 weeks after in-
fection (?1,000 spot-forming cells [SFC] per 106PBMC) with
little apparent decay at 9 weeks postinoculation (Fig. 5A).
Lower-level responses to Gag were observed in the RRV-
seropositive animals, and they decreased at 9 weeks postinoc-
ulation. Clearly positive, although low-level, responses to Tat,
Rev, and Env were also observed, which were higher in RRV-
seropositive animals, while no significant responses to Nef
were observed in either group of vaccinated animals. The low-
est ELISPOT responses were observed in an RRV-seroposi-
tive monkey (440-92), but the other RRV-seropositive mon-
key, 247-04, showed robust responses to Gag and detectable
responses to Tat and Env, as well (Fig. 5A).
Analysis of SIV-specific CD8?T cell responses using MHC
tetramers to the immunodominant Mamu A*01-restricted
Gag181-189CM9 (4) and Tat28-35SL8 (3, 38) epitopes revealed a
similar pattern of immune response after inoculation with re-
combinant RRV. Representative flow cytometric data for
Mamu-A*01 Gag CM9 tetramer binding cells are shown in Fig.
6 for the RRV-naive animal 166-91. A low frequency (0.25% of
CD8?T cells) of Gag CM9 tetramer binding cells was first
observed at 2 weeks postinoculation, rapidly increased to ?9%
by 3 weeks postinoculation, and was sustained at relatively high
levels thereafter until the time of challenge at week 18 (Fig.
7A). A similar pattern was observed in the other RRV-naive
animal, 175-91. These sustained high-level Gag-specific re-
sponses in RRV-naive animals with only minor decreases over
periods of ?10 weeks are strongly suggestive of ongoing anti-
genic stimulation. Gag-specific tetramer responses in RRV-
seropositive animals had lower peaks and, in animal 247-04, a
more accentuated decay. A similar pattern was observed for
Tat SL8-specific responses in both RRV-naïve and RRV-se-
FIG. 7. Summary of Gag CM9 and Tat SL8 tetramer binding cells in RRV-inoculated animals. Macaques 166-91 and 175-91 were RRV naive;
macaques 440-92 and 247-04 were RRV seropositive. Macaques 390-93 and 328-04 were unvaccinated controls that were inoculated with SIV at
week 19. All of these macaques expressed the Mamu-A*01 allele. ?, monkeys that were already RRV positive at the time of RRV-SIV vaccination.
VOL. 85, 2011 VACCINE PROTECTION AGAINST SIV IN MONKEYS12715
ropositive animals, although the overall frequencies of Tat-
specific responses in RRV-naive and RRV-seropositive ani-
mals were lower than for Gag-specific cells (Fig. 7B).
One of the distinguishing characteristics of persistent viral
infections is their ability to induce long-lived effector memory
CD8?T cell responses (23). Phenotypic analysis using Gag and
Tat tetramer binding cells in RRV-vaccinated animals re-
vealed a characteristic effector memory phenotype. Analysis of
both Gag and Tat tetramer binding cells at 5 weeks after
inoculation revealed that SIV-specific cells were predomi-
nantly CD28?CCR7?CD127?and perforin positive, with no
significant evolution between 5 and 12 weeks (Fig. 8A and B).
Serum samples taken on the DOV and over the subsequent
weeks were used to evaluate antibody responses to RRV by
whole-lysed-virus ELISA (Fig. 9). The two RRV-positive mon-
keys, 440-92 and 247-04, as expected, already had strong anti-
body reactivity to RRV proteins on the DOV. The other three
test monkeys (166-91, 175-91, and 128-04) and the RRV-inoc-
ulated control monkey for this study (309-04), as well as one
additional control (232-04), also as expected were RRV neg-
ative on the DOV, but all convincingly seroconverted to strong
anti-RRV antibody reactivity over the subsequent weeks (Fig.
9A). Analysis of serial dilutions of serum demonstrated that by
12 weeks the six seroconverting monkeys had achieved anti-
RRV antibody titers similar to those of monkeys 440-92 and
247-04, which were naturally infected with RRV (Fig. 9B).
Despite the ready detection of SIV Env protein in tissue
culture cells infected with the RRV-SIVenv recombinant and
despite the strong take of RRV-SIV when inoculated into the
RRV-negative monkeys, we were unable to detect anti-Env
antibody responses in the inoculated monkeys. The methods
used included standard and high-sensitivity ELISA to gp120,
Western blotting, and the appearance of neutralizing activity in
serum to the very neutralization-sensitive laboratory-adapted
Challenge phase. At 18 weeks, the five immunized monkeys
and the three immunized controls were challenged intrave-
nously with 10 infectious doses of cloned SIVmac239. The
preparation and titration of this stock has been described pre-
viously (40), and it has been used extensively by a number of
different laboratories for controlled-dose challenges (11, 20,
21, 29, 41, 47). On the basis of SIV RNA burdens in plasma
(Fig. 10) and seroconversion (Fig. 11), all 5 immunized mon-
keys and all 3 controls became infected with SIV.
Viral loads in vaccinated monkeys at peak height (0.49 ? 106
copies of viral RNA per ml of plasma) were 1.85 log10units
lower than those of control monkeys (35.3 ? 106copies of viral
RNA per ml of plasma). This 70-fold (98.6%) reduction was
FIG. 8. SIV-specific CD8?T cells in RRV-vaccinated animals display an effector memory phenotype. (A) Representative flow cytometry data
from monkey 175-91 showing the phenotype of Gag tetramer binding cells (blue) compared with the bulk CD8?T cell population (red) at 12 weeks
postinoculation. (B) Summary of the phenotype of Gag and Tat tetramer binding cells in RRV-vaccinated animals. The frequency of Tat SL8
tetramer binding cells at 12 weeks was too low to provide reliable phenotypic analysis. Samples from animal 440-92 were also excluded due to too
few positive events.
12716 BILELLO ET AL. J. VIROL.
statistically significant using a two-sided two-independent-
group t test for log10(viral loads) (P ? 0.006). Viral loads in
vaccinated monkeys during the chronic phase of infection,
weeks 6 to 32 postchallenge (0.25 ? 105copies of viral RNA
per ml of plasma), were on average 1.80 log10units lower than
those of control monkeys (15.5 ? 105copies of viral RNA per
ml of plasma). This 63-fold (98.4%) reduction was statistically
significant using the Wald test (P ? 0.001) based on a linear
mixed model. Similar findings were obtained when weeks 6
through 12 were analyzed. The viral loads in the control ani-
mals in this study were not significantly different from those in
a number of other studies from a number of different labora-
tories that used the same stock of SIVmac239 (26, 30, 34, 35,
65). The two monkeys that were RRV?at the time of vacci-
nation (440-92 and 247-04) fared no worse than the other
vaccinated monkeys following SIV challenge. There was no
apparent correlation of vaccine phase immune response mea-
surements with postchallenge viral loads.
Analysis of anti-SIVenv antibody responses postchallenge
measured by ELISA revealed responses in all five vaccinated
monkeys and all three control monkeys (Fig. 11 and data not
shown). However, the anti-Env responses in the vaccinated
monkeys rose more quickly and to higher levels than in the
control monkeys, suggestive of an anamnestic response. Cel-
lular responses to SIV antigens also increased to various de-
grees postchallenge on the basis of ELISPOT assays (Fig. 5B
and C) and MHC tetramer binding (Fig. 7).
Except for live attenuated strains of SIV, which have typi-
cally apparent provided apparent sterilizing immunity against
challenge by homologous or closely matched strains, the vast
majority of vaccine challenge experiments in monkeys have not
shown an effect on the acquisition of SIV infection. Protective
effects afforded by vaccination have typically been measured by
the extent to which viral loads have been lowered following
challenge. The extent of viral load reduction afforded by re-
combinant RRV-SIV in the experiments described here com-
pares favorably with maximal viral load reductions that have
been reported in the literature. A number of investigators
using a variety of vaccine approaches have reported maximal
SIV load reductions in the 1.5 to 2.0 log10unit range (geomet-
ric mean) (30, 42, 55, 65). Of course, many studies have not
achieved this degree of viral load reduction. Perhaps the great-
est SIV load reductions have been observed recently by Man-
rique et al. (44). Using recombinant MVA (modified vaccinia
virus Ankara, a poxvirus) expressing SIV Gag, Pol, and Env
proteins, augmented by DNA-mediated cytokine expression,
these investigators reported an approximate 3 log10unit reduc-
tion in viral loads following repeated low-dose mucosal chal-
lenge with SIVmac251 (44).
Hansen et al. (26) have described the construction and per-
formance of replication-competent CMV-SIV recombinants
using the betaherpesvirus from rhesus monkeys. Recombinants
expressing Gag, Env, and a Rev-Tat-Nef fusion protein were
used, as we did here for our RRV-SIV recombinants. More
recently, subsequent to the original submission of our manu-
script, Hansen et al. published a more detailed account of
immune responses and protective efficacy with their rhCMV-
SIV recombinants (25). Interestingly, despite adequate expres-
sion of Env in cells infected in culture, few or no anti-Env
antibody responses were observed by Hansen et al. (26), sim-
ilar to our experience here with the RRV-SIVenv recombi-
nant. Cellular responses with rhCMV-SIV were persistent and
had an effector memory phenotype, also similar to what we
report here. However, the immunodominant A*01-restricted
Gag-CM9 and Tat-SL8 epitopes were not immunodominant in
the context of rhCMV-SIV immunization. Although significant
reductions in chronic-phase viral loads in rhCMV-SIV-vacci-
nated monkeys were not observed by Hansen et al. (25, 26), a
significant fraction of the vaccinated monkeys did not exhibit
progressive systemic dissemination following repeated SIV-
mac239 mucosal exposure.
Virus-specific memory T cells can be categorized into central
memory and effector memory subsets (39). Classically, central
memory T cells are more quiescent than effector memory cells,
have increased proliferative capacity, and retain the ability to
FIG. 9. Antibody responses to RRV during the vaccine phase. An-
tibodies to RRV were measured by ELISA using whole lysed virions
for detection, as previously described (17). Monkeys 440-92 and 247-04
were already RRV positive at the time of enrollment. The straight red
line without data points represents the reactivity of a positive control
(140-83) at a straight 1:10 dilution of serum. The black dashed line
represents the reactivity of a negative control (288-94) at a straight
1:10 dilution of serum. (A) A straight 1:10 dilution of serum was used
at the indicated weeks after vaccine administration. (B) Serial dilutions
of serum taken at week 12 after vaccine administration were tested for
reactivity to RRV virions. ?, monkeys that were already RRV positive
at the time of RRV-SIV vaccination.
VOL. 85, 2011 VACCINE PROTECTION AGAINST SIV IN MONKEYS12717
secrete interleukin 2 (IL-2). Characteristic expression markers
of central memory cells include CD28, CCR7, and CD127.
Central memory T cells are enriched in secondary lymphoid
tissues and relatively absent from mucosal effector sites. In
contrast, effector memory cells are CD28?CCR7?CD127?,
generally express large amounts of perforin, and are highly
enriched in effector mucosal sites. Hansen et al. reported pref-
erential induction of SIV-specific effector memory CD8?T
FIG. 10. SIV RNA loads in plasma following challenge. ?, monkeys that were already RRV positive at the time of RRV-SIV vaccination.
FIG. 11. Antibody responses to SIV gp120 following SIV challenge. Antibodies were measured by ELISA at a straight 1:200 dilution of serum.
?, monkeys that were already RRV positive at the time of RRV-SIV vaccination.
12718BILELLO ET AL.J. VIROL.
cells by their recombinant CMV-SIV strains (26). Although
there was some animal-to-animal variation in our study, SIV-
specific CD8?T cells induced by recombinant RRV similarly
were predominantly a classic effector memory phenotype
(CD28?CCR7?CD127?and perforin positive).
A number of factors could potentially contribute to the poor
elicitation of anti-Env antibodies in our study. Despite the
occurrence of very high anti-Env antibody levels during the
course of HIV-1 and SIV infections (7), the Env protein may
actually not be that immunogenic (37). The high level of anti-
Env antibody responses in natural infection may have more to
do with the prolonged, continuous exposure to high levels of
antigen than it does with the inherent immunogenicity of the
protein. Envelope protein is covered with a glycan shield and is
locked into a tight trimer configuration; others have noticed a
low inherent immunogenicity of the natural gp160 Env protein
when expressed from DNA or other forms of vectored expres-
sion (30, 36, 43, 66). It is also possible that the temporally
unregulated expression of Env in the construct used may be
problematic for the recombinant virus in terms of replication
kinetics, yield per cell, toxicity, or ability to be limited by the
host immune response. Additionally, we do not know whether
Env traffics properly to the cell surface in an RRV-infected cell
that is making a variety of other RRV glycoproteins.
It has been speculated that optimal levels of vaccine protec-
tion may be achieved when both cellular and humoral re-
sponses are optimal and can act in concert (62). Live attenu-
ated strains of SIV persist in monkeys and elicit mature
antibody responses and persistent cellular responses (23, 32).
Our hope in doing these experiments with replication-compe-
tent RRV recombinants was that we could match the charac-
teristics of the anti-SIV immune response achieved by live
attenuated strains of SIV. Although the anti-SIV CD8?cellu-
lar responses that we achieved with RRV-SIV are impressive
in their magnitude and durability, we have certainly failed on
the humoral side. Others have presented evidence for im-
proved efficacy with other vaccine approaches when anti-Env
responses were included in the vaccine regimen (48, 69). If the
problem of anti-Env antibody responses with RRV-SIV can be
resolved, there will still be reason to hope that we can match,
or come close to matching, the degree of protection observed
with live attenuated SIV.
We thank Jennifer Morgan, Hannah Sanford, Jacqueline Gillis, An-
gela Carville, and Mike Piatak for assistance with certain aspects of
these experiments. We also thank David Knipe, George Pavlakis, Da-
vid Watkins, and Eloisa Yuste for providing reagents.
This work was supported by NIH grants R01 AI63928 (R.C.D.),
RR0168 for support of the New England Primate Research Center,
and 5T32AI07245 (J.P.B.). The work was also funded in part with
federal funds from the National Cancer Institute under contract
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