Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys
ABSTRACT The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However,
recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to
protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses
in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys,
divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant
and sustained in vivo activation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher
Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving
the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig
plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent
boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited
humoral and cellular immune responses in higher primates.
- SourceAvailable from: Christopher James Miller[show abstract] [hide abstract]
ABSTRACT: Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Journal of Virology 10/2010; 84(20):10748-64. · 5.08 Impact Factor
Article: Vaccine development against HIV-1[show abstract] [hide abstract]
ABSTRACT: The development of anefficacious vaccine against the human immuno deficiency virus (HIV) is of great urgency, because it is accepted that vaccination is the only means capable of controlling the AIDS pandemic. The foundation of HIV vaccine development is the analysis of immune responses during natural infection and the utilization of this knowledge for the development of protective immunization strategies. Initial vaccine development and experimentation are usually in animal models, including murine, feline, and nonhuman primates. Experimental vaccine can didates are closely studies for both efficacy and safety before proceeding to human clinical trials. There are anumber of different therapeutic and prophylactice vaccine strategies currently being studied in human clinical trials. Vaccine strategies that are being tested, or have previously been te sted, in humans include subunit, DNA plasmid, and viral vector, and combinations of these various strategies. Some of the results of these trials are promising, and additional research has focused on the development of appropriate chemical and genetic adjuvants as well as methods of vaccine delivery to improve the host immune response. This review summarizes the vaccine strategies that have been tested in both animal models and human clinical trials.Immunologic Research 04/2012; 25(1):53-74. · 2.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We investigate the association of different measures of adiposity (waist circumference, hip circumference, waist-to-hip ratio and body mass index) with overactive bladder (urinary frequency and urgency), whether the association varies by gender or age and whether it persists when models are adjusted for other confounders. Data were from the Boston Area Community Health epidemiological survey, a random sample of 5,503 Boston, Massachusetts, residents 30 to 79 years old with equal representation from 3 racial/ethnic groups (black, Hispanic and white). Statistical analysis involved nonparametric loess models and multivariate logistic regression. We noted distinct patterns by gender for the association of various adiposity measures with overactive bladder. Waist-to-hip ratio was not significantly associated with overactive bladder in either gender. In women the prevalence of overactive bladder increased as waist (OR adjusted for other confounders 1.10/10 cm increase) or hip circumference (OR 1.12/10 cm increase) or body mass index (OR 1.03/kg/m2 increase) increased. In men the prevalence of overactive bladder decreased as adiposity increased (OR 0.65/10 cm increase in waist circumference, OR 0.71/10 cm increase in hip circumference and OR 0.87/kg/m2 in body mass index) but only to a certain point (waist circumference 100 cm, hip circumference 115 cm and body mass index 27.5 kg/m2, respectively). At that point the prevalence of overactive bladder increased with increasing adiposity (OR 1.19/10 cm increase in waist circumference, OR 1.16/10 cm increase in hip circumference and OR 1.08/kg/m2 in body mass index). The relationship between adiposity and overactive bladder varies by gender.The Journal of urology 03/2011; 185(3):955-63. · 4.02 Impact Factor
Augmentation of immune responses to HIV-1 and
simian immunodeficiency virus DNA vaccines by
IL-2?Ig plasmid administration in rhesus monkeys
Dan H. Barouch†, Abie Craiu†, Marcelo J. Kuroda†, Jo ¨rn E. Schmitz†, Xin Xiao Zheng†, Sampa Santra†, Julie D. Frost†,
Georgia R. Krivulka†, Michelle A. Lifton†, Carroll L. Crabbs‡, Gwendolyn Heidecker§, Helen C. Perry§, Mary-Ellen Davies§,
Hong Xie§, Christine E. Nickerson†, Tavis D. Steenbeke†, Carol I. Lord†, David C. Montefiori¶, Terry B. Strom†,
John W. Shiver§, Mark G. Lewis?, and Norman L. Letvin†**
†Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215;‡Southern Research Institute, 431 Aviation Way,
Frederick, MD 21701;§Merck Research Laboratories, West Point, PA 19486;¶Duke University Medical Center, Durham, NC 27710;
and?Henry M. Jackson Foundation, 1600 East Gude Drive, Rockville, MD 20850
Edited by William E. Paul, National Institutes of Health, Bethesda, MD, and approved December 30, 1999 (received for review September 29, 1999)
The potential utility of plasmid DNA as an HIV-1 vaccination
modality currently is an area of active investigation. However,
will elicit immune responses of sufficient magnitude to protect
against pathogenic AIDS virus challenges. We therefore investi-
gated whether DNA vaccine-elicited immune responses in rhesus
monkeys could be augmented by using either an IL-2?Ig fusion
protein or a plasmid expressing IL-2?Ig. Sixteen monkeys, divided
into four experimental groups, were immunized with (i) sham
plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus
mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and
IL-2?Ig protein, or (iv) these DNA vaccines and IL-2?Ig plasmid. The
administration of both IL-2?Ig protein and IL-2?Ig plasmid induced
a significant and sustained in vivo activation of peripheral T cells in
the vaccinated monkeys. The monkeys that received IL-2?Ig plas-
mid generated 30-fold higher Env-specific antibody titers and
5-fold higher Gag-specific, tetramer-positive CD8? T cell levels
also augmented the vaccine-elicited immune responses, but less
effectively than IL-2?Ig plasmid. Augmentation of the immune
responses by IL-2?Ig was evident after the primary immunization
demonstrate that the administration of IL-2?Ig plasmid can sub-
stantially augment vaccine-elicited humoral and cellular immune
responses in higher primates.
recognition of the limitations of traditional vaccination mo-
dalities for preventing HIV-1 infection has led to the devel-
opment of a number of novel vaccination strategies, including
recombinant live vectors and plasmid DNA (2). Intramuscular
injection of purified plasmid DNA has been shown to transfect
cells in mice (3) and induce antigen-specific antibody and
cytotoxic T lymphocyte (CTL) responses (4–7). In particular,
plasmids encoding HIV-1 and simian immunodeficiency virus
(SIV) proteins have been shown to elicit specific humoral and
cellular immune responses in both mice (8–10) and rhesus
The immune responses elicited by DNA vaccination have
afforded a degree of protection in nonhuman primates against
challenges with nonpathogenic AIDS viruses (17–20), but
these immune responses have not been of a magnitude suffi-
cient to protect against pathogenic viral challenges (21). We
therefore were interested in exploring strategies for augment-
ing DNA vaccine-elicited immune responses. Augmentation of
vaccine-elicited antibody and CTL responses has been dem-
onstrated in mice by using cytokine administration and by
triggering of costimulatory signaling. However, such ap-
he worldwide spread of HIV-1 (1) will be controlled only
by the development of an effective HIV-1 vaccine. The
proaches, to date, have not been applied successfully in
Augmentation of DNA vaccine-elicited immune responses
using plasmid IL-2 has been reported in several murine disease
models (22–25). We sought to build on this observation by
exploring the utility of IL-2?Ig as a vaccine adjuvant. IL-2?Ig is
a fusion protein that has IL-2 functional activity and the advan-
tages of divalent avidity and a long in vivo half-life (26, 27). We
have reported previously that an IL-2?Ig plasmid was able to
augment the antibody and CTL responses elicited by an HIV-1
gp120 DNA vaccine in mice (28). In fact, IL-2?Ig was signifi-
cantly more effective than native IL-2 as a vaccine adjuvant, and
augmentation was most marked when the plasmid cytokine was
delivered 2 days after the DNA vaccine (28). The present study
was performed to evaluate the ability of plasmid-encoded IL-
2?Ig to augment DNA vaccine-elicited HIV-1 and SIV-specific
immune responses in rhesus monkeys.
Materials and Methods
Construction of IL-2?Ig Plasmids. Human IgG2 cDNA was pre-
pared by reverse transcription–PCR (Stratagene) from an
IgG2-expressing myeloma cell line. Human IL-2 and human
IgG2 Fc were amplified by PCR using Pfu polymerase (Strat-
agene) and synthetic primers with engineered BglII and PvuI
restriction sites (Operon Technologies, Alameda, CA) (IL-2
forward, 5?-cgc aga tct atg tac agg atg caa ctc ctg tct tg-3?; IL-2
reverse, 5?-cgc cga tcg gtc agt gtt gag atg atg ctt tg-3?; IgG2 Fc
forward, 5?-cgc cga tcg caa atg ttg tgt cga gtg ccc acc-3?; IgG2
Fc reverse, 5?-cgc aga tct tat cat tta ccc gga gac agg gag agg ctc-
3?). The PCR products were restriction-digested to generate a
BglII?PvuI IL-2 fragment and a PvuI?BglII Ig fragment. The
pV1J and pCMV (Invitrogen) expression vectors were digested
with BglII, and the pV1J-IL-2?Ig and pCMV-IL-2?Ig plasmids
were constructed by a triple ligation using the vector and both
Production of IL-2?Ig Protein. Murine myeloma NS-1 cells were
stably transfected with the human IL-2?Ig fusion gene. One
microgram of linearized pCMV-IL-2?Ig expression plasmid
containing the neomycin resistance gene was added to 107
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CTL, cytotoxic T lymphocyte; SIV, simian immunodeficiency virus; SHIV,
simian HIV; PBL, peripheral blood lymphocytes.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073?pnas.050417697.
Article and publication date are at www.pnas.org?cgi?doi?10.1073?pnas.050417697
April 11, 2000 ?
vol. 97 ?
washed NS-1 cells in PBS and electroporated at 1.5 kV and 3
?F with a Bio-Rad Gene Pulser System. Transfectants were
selected in R10 medium containing 1.5 mg?ml G418 (Gene-
ticin; Life Technologies, Gaithersburg, MD) and cloned twice
by limiting dilution in 96-well plates. Large-scale cultures of
transfected NS-1 cells were grown in UltraDOMA medium
(BioWhittaker) with 1% low IgG-containing FCS (HyClone).
Culture supernatants were filtered through a 0.2-?m filtration
apparatus, and purification of 10 liters was performed by using
a 2-ml protein A-Sepharose column (Amersham Pharmacia) at
a flow rate of 5 ml?min. The column then was washed with
50 ml of PBS, eluted with 0.1 M citrate, pH 4.0, and imme-
diately neutralized with 0.3 vol of 1 M Tris?HCl, pH 8.
Fractions containing protein were pooled and dialyzed exten-
sively against PBS. The final yield of IL-2?Ig fusion protein was
0.5–1.0 mg?liter of culture supernatant. Analysis of the final
IL-2?Ig fusion protein was performed by SDS?PAGE and
gel-filtration HPLC, and activity was measured by an IL-2
ELISA (BioSource International, Camarillo, CA) and cellular
Selection and Vaccination of Monkeys. To select adult rhesus
monkeys (Macaca mulatta) that expressed the Mamu-A*01
MHC class I allele, a PCR-based assay was utilized (29). Briefly,
DNA was extracted from peripheral blood lymphocytes (PBL)
by using a QIAmp Blood Kit (Qiagen, Chatsworth, CA). PCR
then was performed using Mamu-A*01-specific primers (for-
ward, 5? gac agc gac gcc gcg agc caa 3?; reverse, 5? cgc tgc agc gtc
tcc ttc ccc 3?). Two additional primers specific for a conserved
MHC class II sequence also were included as internal positive
controls (forward, 5? gcc tcg agt gtc ccc cca gca cgt ttc 3?; reverse,
5? gca agc ttt cac ctc gcc gct g 3?). Electrophoresis on a 2%
agarose gel yielded a 685-bp band in Mamu-A*01-positive sam-
ples and a 260-bp control band in all samples. Verification of
positive samples was achieved by complete DNA sequence
analysis and comparison with the published Mamu-A*01 se-
quence (30). Monkeys were housed at Southern Research In-
stitute, Frederick, MD. The animals were maintained in accor-
dance with Henry M. Jackson Foundation and Harvard Medical
Maxipreparations of plasmids were carried out by alkaline
Twelve monkeys were vaccinated by separate intramuscular
injections of 5 mg of HIV-1 89.6P Env (KB9) DNA and 5 mg of
SIV mac239 Gag DNA in sterile saline without adjuvant. Half
the dose was delivered to each quadriceps muscle, and each
injection was delivered in a 0.5-ml volume by using a needle-free
Biojector apparatus and a no. 3 syringe (Bioject, Portland, OR).
Four additional monkeys received 10 mg of sham plasmid DNA.
Of the 12 vaccinated monkeys, 4 received 5 mg of IL-2?Ig
plasmid on day 2 after DNA vaccination, and 4 received
0.5 mg?day IL-2?Ig protein as twice-daily intramuscular injec-
tions on days 1–14 after DNA vaccination.
CD25 Staining of Lymphocytes. Phycoerythrin–Texas red (ECD)-
labeled anti-human CD8?? (2ST8–5H7; Beckman Coulter),
allophycocyanin (APC)-labeled anti-rhesus monkey CD3
(FN18; gift from D. M. Neville, Jr., National Institutes of Health,
Bethesda, MD), and phycoerythrin (PE)-labeled anti-human
CD25 (M-A251; PharMingen) mAbs were used to stain fresh
PBL from vaccinated rhesus monkeys. Antibodies were added to
100 ?l of whole blood, and red blood cells were lysed by using
an Immunoprep Reagent Q-Prep Workstation (Beckman
Coulter). Stained PBL were washed with 3 ml of PBS, resus-
analyzed by using a Coulter EPICS flow cytometry system.
Anti-Env Antibody ELISA. A direct ELISA was used to measure
plasma titers of anti-gp120 antibodies. Ninety-six-well Maxisorp
ELISA plates (Nunc) were coated overnight at 4°C with 100 ?l
of PBS containing 0.5 ?g?ml HIV-1 Env 89.6 gp140 (gift from
ELISA was carried out at room temperature. After a wash with
PBS containing 0.05% Tween-20, the wells were blocked for 2 hr
with a solution containing 2% BSA (Sigma) and 0.05%
Tween-20 in PBS. Plasma samples were serially diluted in 2%
BSA?0.05% Tween-20 and added to the ELISA wells. After a
1-hr incubation, the plate was washed three times and then
incubated with a 1:3,000 dilution of a peroxidase-conjugated
anti-human IgG ? IgM secondary antibody (The Jackson Lab-
oratory) for 1 hr. The plate was washed three times, developed
with 3,3?,5,5?-tetramethylbenzidine (TMB) (K&P Laboratories,
Gaithersburg, MD), stopped with 1% HCl, and analyzed at 450
nm with a Dynatech MR5000 ELISA plate reader.
Functional CTL Assays. PBL from rhesus monkeys expressing the
Mamu-A*01 MHC class I allele (30) were isolated and washed
in Hanks’ balanced salt solution containing 2% FCS. PBL (5 ?
106) in 2 ml of RPMI 1640 medium containing 12% FCS (R12)
were cultured in the presence of 10 ?g?ml optimal p11C peptide
from SIV Gag (amino acids 181–189; CTPYDINQM) (30, 31).
This Mamu-A*01-restricted nonamer peptide previously has
been referred to as p11C, C-M (32). On day 3 of culture, 2 ml
of 40 units?ml human recombinant IL-2 (Hoffman–La Roche)
was added. On day 12 of culture, peptide-stimulated PBL were
centrifuged over Ficoll (Ficoll?Paque) and assessed as effectors
in standard51Cr-release assays by using U-bottomed, 96-well
plates containing 104target cells per well. Autologous B lym-
phoblastoid cell lines pulsed with 1 ?g?ml p11C peptide or p11B
control peptide (ALSEGCTPYDIN) and labeled overnight with
100 ?Ci?ml51Cr were used as targets. All wells were assayed in
duplicate. After incubating effector and target cells together for
4 hr at 37°C, supernatants were harvested, mixed with scintilla-
scintillation counter. To measure spontaneous release of51Cr,
target cells were incubated with 100 ?l of medium, and for
maximum release target cells were incubated with 100 ?l of 2%
Triton X-100. Percent lysis was calculated as: (experimental
release ? spontaneous release)?(maximum release ? sponta-
Tetramer Staining of Peptide-Specific CD8? T Cells. Soluble tet-
rameric Mamu-A*01?p11C complexes were prepared as de-
scribed (32, 33). One microgram of phycoerythrin-labeled tet-
rameric Mamu-A*01?p11C complexes in conjunction with
FITC-labeled anti-human CD8? (Leu2a; Becton Dickinson),
ECD-labeled anti-human CD8?? (2ST8–5H7; Beckman
Coulter), and APC-labeled anti-rhesus CD3 (FN18) mAbs was
used to stain p11C-specific CD8? T cells as described (32). One
hundred microliters of whole blood from the vaccinated mon-
keys was directly stained with these reagents, lysed on an
Immunoprep Reagent Q-Prep Workstation (Coulter), washed in
3 ml of PBS, and fixed in 0.5 ml of PBS containing 1.5%
paraformaldehyde. Alternatively, 5 ? 105cultured PBL from the
12-day, peptide-stimulated cultures were similarly stained,
washed, and fixed. Samples were analyzed by four-color flow
cytometry on a Coulter EPICS Elite ESP system. Gated
CD3?CD8? T cells were examined for staining with tetrameric
Statistical Analysis. To determine whether immune responses
among the groups of monkeys were significantly different from
each other, ANOVA was performed by using a 95% confidence
interval (Microsoft EXCEL 97 statistical package). A value of P ?
0.05 was considered significant.
Barouch et al.
April 11, 2000 ?
vol. 97 ?
no. 8 ?
Production of IL-2?Ig Plasmid and IL-2?Ig Protein. The cytokine?Ig
fusion protein selected for evaluation in this study consisted of
human IL-2 fused to the Fc portion of human IgG2. We utilized
a construct based on human sequences because rhesus monkey
and because human IL-2 is capable of driving the proliferation
of rhesus monkey T lymphocytes. The IgG2 isotype was chosen
because of its limited capacity to facilitate antibody-dependent,
cell-mediated cytotoxicity and complement fixation. The plas-
mid-encoded IL-2?Ig used in this study was a pV1J vector (13,
34, 35) expressing IL-2?Ig under control of a strong cytomega-
lovirus (CMV) promoter and enhancer. Expression was con-
firmed by transient transfection of COS cells followed by analysis
of culture supernatants by using an IL-2 ELISA (data not
shown). To produce IL-2?Ig fusion protein, a pCMV vector
expressing IL-2?Ig was constructed and used to transfect murine
NS-1 myeloma cells as described (36). Stable transfectants were
selected, cloned, and screened for high levels of IL-2?Ig expres-
sion. The IL-2?Ig protein then was purified from large volumes
of tissue culture supernatant by using protein A-Sepharose
columns. The purified IL-2?Ig protein was a single, 45-kDa band
as analyzed by SDS?PAGE, a 90-kDa dimer as determined by
gel-filtration chromatography, readily detected by using an IL-2
ELISA, and fully biologically active as determined by CTLL
proliferation assays (data not shown). The endotoxin level of the
final product was less than 10 units?ml (Sigma E-Toxate assay).
Vaccine Trial Design.SIV Gag-specific CTL in rhesus monkeys can
through analysis of CD8? T lymphocyte recognition of the
optimal Gag p11C epitope (amino acids 181–189) restricted by
the HLA-A homolog allele Mamu-A*01 (29, 30). A colony of
rhesus monkeys therefore was screened for the presence of the
Mamu-A*01 gene by PCR using Mamu-A*01-specific primers.
Eleven Mamu-A*01-positive monkeys were identified by PCR
and confirmatory DNA sequencing. These monkeys, as well as
5 Mamu-A*01-negative monkeys, were included in the vaccina-
DNA vaccines expressing HIV-1 89.6P Env gp140 (KB9) or
SIV mac239 Gag in the pV1R backbone were used as immu-
nogens. The HIV-1 89.6 envelope gene, which was derived from
a primary patient R5?X4 dual-tropic HIV-1 isolate, was used
with the SIVmac239 backbone to construct a simian-HIV
(SHIV) chimera (37). In vivo passage of this virus yielded the
pathogenic virus SHIV89.6P. The KB9 envelope clone was
derived from this pathogenic viral isolate (38).
Four groups of rhesus monkeys, each composed of four
animals, were vaccinated as outlined in Fig. 1. The 11 available
Mamu-A*01-positive monkeys were placed into the groups re-
ceiving the experimental DNA vaccines. Ten milligrams of a
sham plasmid was injected into the quadriceps muscles of one
group of rhesus monkeys by using a needle-free Biojector
apparatus. Five milligrams of the env DNA vaccine and 5 mg of
the gag DNA vaccine were administered as separate injections to
the other three groups by the same method of inoculation. Of
these vaccinated animals, one group also received 5 mg of sham
plasmid on day 2 after vaccination, one group received 5 mg of
IL-2?Ig plasmid on day 2 after vaccination, and one group
received 0.5 mg?day IL-2?Ig protein on days 1–14 after vacci-
nation. The IL-2?Ig protein was injected intramuscularly in two
divided doses per day. The monkeys received a similar immu-
nization with cytokine administration at week 4 and another
immunization without cytokine administration at week 8. The
vaccination and cytokine regimens were well tolerated.
Plasma IL-2?Ig levels in the vaccinated animals were deter-
mined by ELISA. Peak levels, measured 3 hr after the first
IL-2?Ig protein injection, ranged from 41 to 102 ng?ml. Trough
levels ranged from 13 to 23 ng?ml. Comparable trough cytokine
levels were detected on days 5 and 14, suggesting that no
functionally significant neutralizing antibody response against
the cytokine fusion protein developed during this period of
cytokine administration. Plasma IL-2?Ig levels in the animals
that received the IL-2?Ig plasmid or in the animals that received
the DNA vaccines alone were ?0.1 ng?ml.
CD25 Expression on Peripheral T Cells of the Vaccinated Monkeys.
CD25 expression on peripheral T cells served as a marker for
activation and in vivo responsiveness to IL-2?Ig. As shown in Fig.
2, CD3? lymphocytes from the animals that received the DNA
vaccines alone did not demonstrate significant levels of CD25
expression. In contrast, CD3? lymphocytes from the animals
imental groups and were immunized with: 1) 10 mg of sham plasmid; 2) 5 mg
and IL-2?Ig protein; or 4) DNA vaccines and IL-2?Ig plasmid. Five milligrams of
alone on day 2 after the week 0 and week 4 immunizations. IL-2?Ig protein
the week 0 and week 4 immunizations. Five milligrams of IL-2?Ig plasmid was
administered on day 2 after the week 0 and week 4 immunizations. The week
8 immunization was performed without cytokine administration.
Vaccine trial design. Sixteen monkeys were divided into four exper-
CD25 expression on CD3? PBL of the vaccinated monkeys. The monkeys were
bled at week 0, week 1, week 2, and every 2 weeks thereafter. Gated CD3?
mAb by flow cytometry. The monkeys that received IL-2?Ig protein demon-
strated a striking rise and rapid fall of CD25 expression. The monkeys that
received IL-2?Ig plasmid showed a less dramatic but more sustained increase
in CD25 expression. Means and SE for each group are shown.
IL-2?Ig protein and IL-2?Ig plasmid administration lead to significant
www.pnas.orgBarouch et al.
that received IL-2?Ig protein showed a dramatic increase in
CD25 expression during the time of cytokine?Ig treatment,
peaking at 1 week after the first inoculation and then declining.
The percentage of CD3? lymphocytes that expressed CD25 in
the monkeys that received IL-2?Ig plasmid was initially low, but
the level of CD25 expression in these monkeys remained signif-
icant and sustained through week 12 at a level comparable to the
T cells of the monkeys that received IL-2?Ig protein. CD25
expression on CD4? T cells and CD8? T cells showed similar
kinetic trends, although slightly higher levels of CD25 expression
were evident on CD4? T cells and slightly lower levels on CD8?
T cells (data not shown).
Humoral Immune Responses. Plasma samples from the vaccinated
monkeys were analyzed for anti-Env antibodies by a direct
ELISA using purified Env 89.6 protein. Fig. 3 shows that
anti-Env antibodies were detected in the vaccinated monkeys by
week 2 after primary immunization. All the DNA-vaccinated
animals seroconverted by week 4 and had higher anti-Env
antibody titers after the boost immunizations. In contrast, the
sham-vaccinated animals did not develop detectable anti-Env
antibodies. By week 12, the group that received IL-2?Ig protein
had a 10-fold higher antibody titer when compared with the
group that received IL-2?Ig plasmid had more than a 30-fold
higher antibody titer when compared with the group that
received the DNA vaccines alone (P ? 0.001). These antibodies
were not capable of neutralizing free virus.
Cellular Immune Responses. Staining with tetrameric MHC class
I?peptide complexes and analysis by flow cytometry recently has
proven to be an accurate method for quantitating epitope-
specific CTL in both whole-blood and peptide-stimulated cell
cultures (32, 33). Both tetramer staining and functional cyto-
toxicity assays documented the rapid emergence of SIV Gag-
specific CTL in the vaccinated animals. CTL specific for the
(CTPYDINQM) (30, 31) were monitored in the 11 Mamu-A*01-
positive monkeys by tetramer staining of fresh PBL, tetramer
staining of PBL stimulated in vitro with the p11C peptide, and
functional51Cr release assays using peptide-stimulated PBL as
effector cells. Fig. 4 shows an example of the tetramer staining
and functional lysis data generated in monkey 772, a represen-
tative animal that received the DNA vaccines and IL-2?Ig
protein. Tetramer-positive CD3?CD8? cells were detected in
peptide-stimulated PBL 2 weeks after the primary immunization
and in freshly isolated PBL 2 weeks after the second immuni-
zation. The level of functional cytotoxicity correlated well with
the level of tetramer staining in these lymphocyte populations.
These data suggest that the tetramer-positive CD3?CD8? cells
represent functional CTL.
Fig. 5 summarizes the tetramer staining and functional cyto-
toxicity data of p11C-stimulated PBL from the Mamu-A*01-
p11C-specific CTL were detected in the preimmune bleeds from
the monkeys. By week 4, tetramer-positive CD8? T cells and
functional CTL were detected in peptide-stimulated PBL from
all the vaccinated monkeys. Background tetramer staining of the
week 4 peptide-stimulated PBL using a control Mamu-A*01?Pol
p68A (STPPLVRLV) tetramer was ?0.1% (data not shown).
Fig. 5A depicts the mean percentage of Mamu-A*01?Gag p11C
tetramer staining of p11C-stimulated PBL for each group of
monkeys, demonstrating that IL-2?Ig plasmid administration
significantly augmented the Gag p11C-specific CD8? T cell
response and that IL-2?Ig protein administration had less of an
enhancing effect. Fig. 5B shows that the mean functional cyto-
elicited anti-Env antibody responses in monkeys. Plasma was obtained from
ELISA. Sham-vaccinated monkeys did not have detectable anti-Env antibody
titers. The most striking and consistent augmentation of anti-Env antibody
titers was observed in the animals that received IL-2?Ig plasmid. Geometric
mean titers and SE for each group are shown.
IL-2?Ig protein and IL-2?Ig plasmid administration augment vaccine-
tetramer staining and functional cytotoxicity assays in a representative
vaccinated monkey. Tetramer staining of fresh PBL, tetramer staining of
p11C-stimulated PBL, and functional lysis of p11C-pulsed targets by
epitope peptide-stimulated PBL are shown for the following time points:
week 0 (preimmune), week 2 (2 weeks after the primary immunization),
week 6 (2 weeks after the second immunization), and week 10 (2 weeks
after the third immunization). Staining with the Mamu-A*01?p11C tet-
ramer was assessed by four-color flow cytometry on gated CD3?CD8?
lymphocytes. Functional lysis was determined by using p11C epitope pep-
tide- and p11B control peptide-pulsed target cells. Tetramer staining was
evident in peptide-stimulated PBL after the primary immunization and in
fresh PBL after the second immunization. Functional lysis correlated well
with tetramer staining. This particular monkey (772) received the DNA
vaccines and IL-2?Ig protein.
Evolution of the SIV Gag p11C-specific CTL response detected by
Barouch et al.
April 11, 2000 ?
vol. 97 ?
no. 8 ?
toxicity of these peptide-stimulated PBL correlated with the
levels of tetramer staining.
The IL-2?Ig-mediated augmentation of the p11C-specific
CTL responses persisted after the boost immunizations in the
Mamu-A*01-positive monkeys, as shown in Fig. 6. Tetramer-
positive CD8? T cells were detected in freshly isolated PBL by
week 6, 2 weeks after the second immunization. The animals
that received IL-2?Ig plasmid had significantly higher levels of
circulating tetramer-positive CD8? T cells as compared with
the animals that received DNA vaccines alone (P ? 0.05). The
tetramer-positive CD8? T cell responses peaked 2 weeks after
each boost immunization and then declined. At week 10, 2
weeks after the third immunization, p11C-specific CD8? T
cells accounted for an average of 0.6% of circulating T cells in
both groups of monkeys that received IL-2?Ig, a level 5-fold
higher than in the animals that received the DNA vaccines
Cytokines have been shown in a number of murine models to
enhance virus-specific immune responses elicited by DNA vac-
cines. Coinoculation of a plasmid expressing granulocyte?
macrophage colony-stimulating factor (GM-CSF) with a rabies
virus DNA vaccine was demonstrated to increase the rabies-
specific antibody response in mice (39). Administration of
plasmids expressing IL-2 or GM-CSF also has been reported to
C virus DNA vaccines (22–24). We previously have reported that
an IL-2?Ig fusion protein can augment DNA vaccine-elicited
immune responses in mice more effectively than native IL-2 (28).
The present study demonstrates clear-cut, enhanced, vaccine-
elicited humoral and cellular immune responses through cyto-
kine administration in nonhuman primates. The augmentation
of tetramer-positive CD8? T cell responses was observed 2
weeks after vaccination and persisted over time for at least 32
We have shown effectiveness of cytokine administration in
monkeys by using both IL-2?Ig protein and IL-2?Ig plasmid.
Because IL-2 has been shown in vitro to activate T cells and to
induce the expression of the IL-2 receptor subunit CD25, we
monitored these effects in vivo on circulating T lymphocytes in
the monkeys that received IL-2?Ig. Administration of either
IL-2?Ig protein or IL-2?Ig plasmid led to a significantly in-
both methods of cytokine administration induced IL-2 receptor
stimulation. The animals treated with IL-2?Ig protein showed a
dramatic rise followed by a rapid fall in CD25 expression,
whereas those injected with IL-2?Ig plasmid showed a lower but
sustained level of CD25 expression. Interestingly, by week 8, the
two groups of IL-2?Ig-treated animals had comparable levels of
CD25 expression on peripheral T cells. Because the monkeys
that received the IL-2/Ig plasmid had undetectable levels of
plasma IL-2?Ig (?0.1 ng?ml), the IL-2?Ig plasmid likely func-
tions by expressing a low but persistent level of functional
cytokine in vivo.
The temporal sequence of administering the IL-2?Ig plasmid
after the DNA vaccine in the present study was based on a
protocol devised in our previous experiments optimizing the
delivery of these constructs in mice (28). We have not carried out
an analogous study to optimize such a protocol in monkeys. In
the present study, the IL-2?Ig plasmid was more effective than
the IL-2?Ig protein in augmenting vaccine-elicited humoral and
cellular immune responses, suggesting that a sustained release of
low-dose IL-2 may be more effective than shorter courses of
high-dose IL-2 as a vaccine adjuvant. In addition, administering
single injections of IL-2?Ig plasmid has significant practical and
economic advantages over frequent dosing of purified IL-2?Ig
protein. The IL-2?Ig plasmid also may prove useful in augment-
ing immune responses elicited by other vaccine modalities.
We acknowledge Meryl A. Forman, Robert W. Doms, Keith A.
Reimann, Wenyu Lin, and Frederick Vogel for generous advice, assis-
tance, and reagents. We acknowledge support from Grants AI?GF-
41521 (T.B.S.), AI-42298 (T.B.S.), NO1-AI-65301 (M.G.L.), AI-85343
(N.L.L.), and CA-50139 (N.L.L).
p11C-specific CTL responses in fresh PBL from the vaccinated monkeys. p11C-
PBL. Staining with the Mamu-A*01?p11C tetramer was assessed by four-color
flow cytometry on gated CD3?CD8? lymphocytes. The most consistent and
significant augmentation was observed in the monkeys that received IL-2?Ig
plasmid. Means and SE for each group are shown.
IL-2?Ig protein and IL-2?Ig plasmid administration augment SIV Gag
p11C-specific CTL responses in monkeys after primary immunization. p11C-
specific CTL responses were analyzed every 2 weeks by tetramer staining (A)
and functional cytotoxicity assays (B) using peptide-stimulated PBL. Staining
with the Mamu-A*01?p11C tetramer was assessed by four-color flow cytom-
etry on gated CD3?CD8? lymphocytes. Specific functional lysis was deter-
mined by using chromium-release cytotoxicity assays at effector-to-target
IL-2?Ig protein and IL-2?Ig plasmid administration augment SIV Gag
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