Download A human immunodeficiency virus type 1 Env–granulocyte

Journal
of General Virology (1999), 80, 217–223. Printed in Great Britain
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A human immunodeficiency virus type 1 Env–granulocytemacrophage colony-stimulating factor fusion protein enhances
the cellular immune response to Env in a vaccinia virus-based
vaccine
Dolores Rodrı! guez,1 Juan Ramo! n Rodrı! guez,1 Mercedes Llorente,2 Isabel Va! zquez,1 Pilar Lucas,2
Mariano Esteban,1 Carlos Martı! nez-A.2 and Gustavo del Real2
Departments of Molecular & Cellular Biology1 and Immunology & Oncology2, Centro Nacional de Biotecnologı! a, CSIC/UAM,
Campus de Cantoblanco, E-28049 Madrid, Spain
Vaccinia virus (VV) infection induces protective T- and B-cell responses, making recombinants
based on VV good candidates for the development of effective vaccines to other viruses. VV
recombinants expressing the human immunodeficiency virus (HIV) envelope protein (Env) have
been generated in several laboratories and shown to induce anti-HIV cellular and humoral immune
responses in vaccinated humans and in chimpanzees. To increase the immunogenicity of the Env
antigen, a VV recombinant was generated that expresses a chimeric antigen consisting of the Env
protein fused to an immunostimulatory cytokine, granulocyte-macrophage colony-stimulating
factor (GM-CSF). The chimeric protein retained GM-CSF biological activity when expressed by this
recombinant virus (VV-GM–gp120) in cells infected in vitro. Infection of BALB/c mice with VVGM–gp120 triggered a higher HIV-specific cellular immune response, as measured by interferonγ production, than that induced by a VV recombinant expressing the native Env protein. Moreover,
although anti-gp120 antibody titres were similar in sera from mice inoculated with either of the VV
recombinants, immunization with the recombinant expressing the fusion protein elicited antibodies
against a broader spectrum of Env epitopes. These results indicate that HIV Env antigen fusion to
GM-CSF provides a means to improve the anti-HIV immune response.
Introduction
Vaccinia virus (VV), the best-characterized member of the
family Poxviridae, has proven to be an effective vaccine, as its
use has resulted in the global eradication of smallpox (Fenner
et al., 1988). The possibility of adverse effects after vaccination
with VV has been minimized by inactivation of genes encoding
virulence factors (Moss, 1996 ; Paoletti, 1996). VV recombinants expressing human immunodeficiency virus (HIV) antigens have been developed in several laboratories (Chakrabarty
et al., 1986 ; Hu et al., 1986 ; Rodrı! guez et al., 1989) in order to
investigate their potential utility as vaccines against HIV
infection. Phase I clinical trials in healthy individuals vaccinated
with different HIV-1 envelope protein (Env) poxvirus recombinants demonstrated that such a vaccine is safe and induces
Author for correspondence : Mariano Esteban.
Fax j34 91 585 4506. e-mail mesteban!cnb.uam.es
0001-5728 # 1999 SGM
humoral and T-cell responses specific for this HIV antigen
(Cooney et al., 1993 ; Graham et al., 1993 ; McElrath et al., 1994 ;
Pioloux et al., 1995 ; Egan et al., 1995 ; Fleury et al., 1996). Most
of these reports describe an enhancement of the anti-Env
immune response when individuals primed with a poxvirus
recombinant were boosted with purified recombinant gp160.
These experiments suggest that, although such a strategy
appears promising, a more effective primary response would
be of considerable importance. In an attempt to improve
presentation of HIV antigen to the host immune system, we
have generated a VV recombinant expressing a chimeric HIV
Env–granulocyte-macrophage colony-stimulating factor (GMCSF) fusion protein.
GM-CSF is a haematopoietic factor that induces proliferation of haematopoietic precursor cells and their differentiation to neutrophils, eosinophils and macrophages (Gough
et al., 1984 ; Metcalf, 1986). It is also the main stimulatory
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D. Rodrı! guez and others
cytokine for the viability, differentiation and function of
Langerhans’ and dendritic cells (Sallusto & Lanzavecchia, 1994),
which are the most important cell types for the stimulation in
vivo of primary T-cell-mediated immune responses (Steinman,
1991). GM-CSF also enhances monocyte surface MHC class II
expression, adhesion molecules and co-stimulatory factors, and
stimulates interleukin-1 secretion, critical for antigen presentation to T lymphocytes (Smith et al., 1990). The immunostimulatory activity of GM-CSF permits enhancement of T-cell
proliferative responses to suboptimal antigen concentrations
and an increase in primary antibody responses (Morrissey et al.,
1987 ; Grabstein et al., 1996 ; Disis et al., 1996 ; Kim et al., 1997).
GM-CSF has also been shown to increase tumour immunogenicity in animal models (Dranoff et al., 1993). Cytokinetransduced autologous melanoma cells have been used as a
therapeutic vaccine in humans, resulting in the generation of an
anti-tumour immune response associated with clinical benefit
(Ellem et al., 1997).
Here we report that immunization with a VV recombinant
expressing GM-CSF fused to HIV-1 Env resulted in an
augmented cellular immune response compared with that
elicited by a VV recombinant expressing the Env protein alone.
Both HIV-Env-specific T-cell proliferation and the number of
interferon-γ (IFN-γ)-producing CD8+ cells increased in mice
immunized with the fusion protein, indicating that T-cellmediated immune responses were augmented by the presence
of GM-CSF. Although serum antibody titres were similar in
mice infected with either of the VV recombinants, immunization with the fusion protein elicited antibodies against a
broader spectrum of gp120 epitopes. The combined effect of
the increase in these responses and the more extensive
antibody repertoire triggered by GM-CSF may be critical in
promoting a neutralizing response to HIV-1.
Methods
Cells and viruses. African green monkey kidney cells (BSC-40)
were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % newborn calf serum. P815 mastocytoma cells, which
express MHC class I molecules, were cultured in DMEM containing 10 %
foetal calf serum (FCS). The VV strain WR was propagated and titrated
in BSC-40 cells. The recombinant virus VV-ENV, expressing the entire
env gene of HIV-1 (strain IIIB), has been described previously (Rodrı! guez
et al., 1989). The human lymphoid cell line MT2 was grown in RPMI
supplemented with 10 % FCS.
VV recombinant construction. Generation of the VV insertion
vectors VV-GM-CSF and VV-GM–gp120 is explained in the legend to
Fig. 1. The GM-CSF and GM–gp120 genes were inserted into the
thymidine kinase (TK) region of the VV WR strain by DNA homologous
recombination. β-Galactosidase-producing plaques were picked, cloned
three times and amplified in BSC-40 cells in culture. Recombinant viruses
were purified by banding in sucrose gradients, as previously described
(Rodrı! guez et al., 1989).
Determination of GM-CSF biological activity. The biological
activity of the GM–gp120 fusion protein was assayed by testing its
ability to support proliferation of the murine GM-CSF-dependent cell line
CBI
NFS-60. Cells (10&\ml) were seeded into 96-well plates with dilutions of
supernatants from cell cultures infected with VV-GM–gp120 or infected
with a VV recombinant (VV-GM-CSF) expressing native GM-CSF. After
24 h incubation, 10 ml MTT solution (5 mg\ml 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide in PBS ; Sigma) was added and the
plates were incubated for 4 h at 37 mC. The reaction was terminated by
addition of 100 ml SDS\HCl solution (10 % SDS in 10 mM HCl) and the
plates were incubated overnight at 37 mC. Absorbance was measured at
550 nm with a reference wavelength of 690 nm.
Immunization. Six- to eight-week-old female BALB\c mice, bred in
the CNB animal facility, were used. Groups of mice (three per group)
were primed by infection with 5i10( p.f.u. VV inoculated by the
intraperitoneal route. Animals were boosted intraperitoneally 21 days
later with HIV-1 IIIB-derived soluble gp120 (5 µg per animal ; Intracel).
Synthetic peptides. The peptide GPGRAFVTI, an H-2d-restricted
CD8+ T-cell epitope corresponding to the V3 loop of HIV-1 strain IIIB,
was generously provided by Margarita del Val (Inst. Salud Carlos III,
Madrid). A collection of 47 overlapping peptides (20-mers with a 10amino-acid overlap) covering the entire gp120 protein was provided by
the AIDS Reagent Project (Medical Research Council, UK ; repository
reference ADP740\1-47).
ELISA determination of anti-gp120 antibodies in mouse
sera. ELISA microtitre plates (Maxi-sorb, Nunc) were coated by
overnight incubation at 4 mC with purified gp120 (1 µg\ml ; strain IIIB,
Intracel) or peptides (10 µg\ml) in PBS. After blocking with 0n5 % BSA in
PBS, wells were washed with distilled water and incubated for 60 min at
37 mC with diluted mouse sera. Antibody reactivity was determined after
incubation with peroxidase-labelled goat anti-mouse IgG and addition of
O-phenylenediamine dihydrochloride (Sigma). The reaction was terminated with 3 M sulphuric acid and absorbance was determined at 492 nm.
The titre was expressed as the highest serum dilution giving an
absorbance value three times that of the preimmune serum.
Cell proliferation assay. Spleens were removed from infected
mice and single-cell suspensions were prepared in complete medium
(RPMI 1640 with 10 % FCS, 2 mM -glutamine and 10 µM 2mercaptoethanol). Splenocytes (10' per well) were cultured in 96-well
microtitre plates. Cultures were challenged in triplicate with 2 µg\ml
gp120 and incubated for 3 days at 37 mC with 5 % CO , after which 1 µCi
#
[$H]thymidine (5 Ci\mmol ; Amersham) was added to each well. After
16 h, cells were harvested and [$H]thymidine incorporation into DNA
was measured by liquid-scintillation counting. Concanavalin A (1 mg\ml ;
Sigma) was used as a polyclonal stimulator control. The stimulation index
(SI) was determined as the ratio (experimental count – spontaneous
count)\spontaneous count.
ELISPOT assay. The ELISPOT assay was used to detect epitopespecific IFN-γ-secreting cells, as previously described (Miyahira et al.,
1995). Briefly, 96-well nitrocellulose-bottomed plates were coated with
anti-mouse IFN-γ MAb R4-6A2 (8 µg\ml, Pharmingen). After overnight
incubation at room temperature, wells were washed three times with
RPMI 1640 and 100 µl medium supplemented with 10 % FCS was added
to each well. Plates were then incubated at 37 mC for 1 h. Duplicate
cultures were prepared from immunized mouse splenocytes in serial
doubling dilutions, beginning with 10' cells per well. P815 (H-2d) cells
were used as antigen-presenting cells (APC) and were pulsed with 10−'
M synthetic peptide GPGRAFVTI and treated with mitomycin C
(30 µg\ml, Sigma). After several washes with culture medium, 10& P815
cells were added to each well. P815 cells not pulsed with the peptide but
cultured under similar conditions were used as a control. Plates were
incubated for 26–28 h at 37 mC and then washed with PBS containing
0n05 % Tween 20 (PBS-T) before being incubated overnight at 4 mC with
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Immunostimulation by HIV-1 Env–GM-CSF
Fig. 1. Scheme for the construction of VV insertion vectors containing the murine GM-CSF coding sequence or the GM–gp120
chimeric gene. A DNA fragment containing the GM-CSF gene was excised from plasmid pBacPAKGM-CSF by restriction with
XhoI and EcoRI. This fragment was either cloned directly into the SmaI site of the VV insertion vector pSC11, generating pSCGM-CSF, or digested with SpeI to eliminate the GM-CSF stop codon (denoted by an asterisk) and cloned into plasmid
pBacPAK-gp120, which had been previously digested with XhoI and SpeI to eliminate the gp120 signal peptide (SP). The
chimeric gene was excised by digestion of the resulting plasmid, pBacPAKGM–gp120, with XhoI and EcoRI, treated with
Klenow and cloned into pSC11 to generate the pSC-GM–gp120 VV transfer vector. The GM-CSF gene and the GM–gp120
chimeric sequence were introduced into the thymidine kinase (TK) locus of the VV genome after transfection of VV-infected
cells with the plasmids pSC-GM-CSF and pSC-GM–gp120, respectively.
(b)
5
1.0
gp160
gp120
0.8
0.6
0.4
Results
0.2
Analysis of protein expression by recombinant VV
0.0
The strategy for generation of VV recombinants expressing
GM-CSF and the GM–gp120 fusion protein is outlined in Fig.
1. To demonstrate that VV-GM–gp120 expressed the fusion
protein, BSC-40 cells were infected with VV-GM-CSF, VVGM–gp120 or VV-ENV, cell extracts were separated by
SDS–PAGE and Western blots were probed with a polyclonal
anti-gp120 antibody (ADP 422, MRC AIDS Reagent Project).
In VV-ENV-infected cells, the anti-gp120 antibody reacted
with the Env precursor gp160 and the gp120 processed form
(Fig. 2 a, lane 5) but in cells infected with different VVGM–gp120 isolates (lanes 2–4), the antibody reacted with a
protein with an apparent molecular mass of 150 kDa. In an
ELISA in which the GM-CSF portion of the fusion protein was
captured by a rat anti-GM-CSF antibody (Endogen), the gp120
moiety was revealed with two different monoclonal antibodies
(F7, an anti-V2 produced in our laboratory and NEA-9284, an
anti-V3 from Dupont) and two rabbit polyclonal anti-gp120
antisera (ADP 422 and CNB.1, produced in our laboratory),
VV-GM-CSF
VV-GM– gp120
VV-ENV
1/100
1/200
1/400
1/800
1/1600
1/3200
1/6400
1/12800
1/25600
1/51200
(a)
1 2 3 4
A550
biotinylated anti-mouse IFN-γ MAb XMG1.2 (2 µg\ml, Pharmingen) in
PBS-T. Plates were then washed with PBS-T and 100 µl 1\800-diluted
peroxidase-labelled avidin (Sigma) in PBS-T was added to each well.
Wells were washed 1 h later with PBS-T and PBS. The spots were
developed by adding 50 mM Tris–HCl (pH 7n5) containing 1 µg\ml
3,3h-diaminobenzidine tetrahydrochloride (Sigma) and 0n015 % H O .
# #
When the plates were completely dry, the number of spots was
determined with the aid of a stereo-microscope.
Supernatant dilution
Fig. 2. Expression of biologically active GM–gp120 fusion protein in VVGM–gp120-infected cells. (a) Western blot analysis of BSC-40 cells
infected (5 p.f.u. per cell) with VV-GM–gp120 or a recombinant virus, VVENV, expressing the entire HIV-1 Env protein. Cell extracts prepared at
24 h post-infection were fractionated on 10 % SDS–PAGE gels, transferred
to nitrocellulose and incubated with gp120-specific rabbit polyclonal
antibodies. Lane 1, extract from cells infected with wild-type VV ;
2–4, extracts from cells infected with VV-GM–gp120 isolates ; 5, extract
from cells infected with VV-ENV. (b) Biological activity of GM-CSF and the
GM–gp120 recombinant protein. Supernatants from BSC-40 cells infected
for 24 h with VV-GM-CSF or VV-GM–gp120 recombinants were tested as
a source of GM-CSF, which allows proliferation of the murine GM-CSFdependent NFS-60 cell line. Proliferation was quantified as described in
Methods.
indicating that the antigen was properly expressed (data not
shown). The biological activity of GM-CSF and the chimeric
GM–gp120 protein expressed by the VV recombinants was
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Anti-gp120 IgG titre (× 10 – 4)
D. Rodrı! guez and others
4
(a)
3.5
3
2.5
2
1.5
1
0.5
0
(b)
VV-GM-CSF
VV-GM–gp120
VV-ENV
VV-GM –gp120
VV-ENV
2.5
1.6
1.4
2
1.2
A492
A492
1
0.8
0.6
0.4
0.2
0
2.5
1.5
1
0.5
1.2
1
2
A492
A492
0.8
1.5
0.4
0.5
0.2
2.5
1.6
1.4
2
1.2
1.5
1
Fig. 3. Humoral
0.8 anti-HIV-1 gp120 immune response elicited in mice
immunized with VV-Env recombinants. Groups of mice (three per group)
were primed 0.6
by vaccination (5i107 p.f.u.) with recombinants VV-ENV,
VV-GM–gp120
0.4or VV-GM-CSF. Mice were boosted 21 days later with
soluble gp120 (5 µg per mouse) and serum samples were collected 29
days later. (a)0.2Anti-gp120 reactivity in pooled serum samples was
analysed by ELISA with recombinant gp120-coated plates, as described
in Methods. (b) Epitope mapping of mouse sera. Sera from three
individual mice immunized with VV-ENV or VV-GM–gp120 were tested
by ELISA for reactivity against a series of overlapping synthetic peptides
gp 120 overlapping peptides
covering the entire HIV Env protein.
A492
A492
1
1
0.5
gp 120 overlapping peptides
determined using a GM-CSF-dependent cell line. Cultured cells
infected with either of the recombinant viruses, expressing the
native or chimeric GM-CSF proteins, supported the growth of
the dependent cell line, although the chimeric protein was
approximately 50 % less efficient (Fig. 2 b). By FACS analysis
we have demonstrated binding of the gp120 domain of the
chimeric protein to CD4-bearing human cells (MT2) and this
binding was specific, as it could be blocked with soluble CD4
(ADP 609, MRC AIDS Reagent Project) (data not shown).
These findings show that the VV-GM-CSF recombinant
CCA
0.6
produces a biologically active GM-CSF molecule and that the
VV-GM–gp120 recombinant expresses a protein of approximately 150 kDa that exerts GM-CSF activity, binds to human
CD4 receptor and is recognized by poly- and monoclonal antigp120 antibodies.
Humoral immune response elicited by the HIV-1 Env
recombinants
We next tested the extent of the humoral and cellular
immune responses induced in mice vaccinated with the VV
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Immunostimulation by HIV-1 Env–GM-CSF
Chimeric GM–Env enhances the specific cellular
immune response to gp120
T-helper-cell activation and proliferation is essential in
inducing both humoral immune responses via B cells and
cellular immune responses via CD8+ cytotoxic T cells. It was
thus of interest to determine the extent of the HIV-1-specific Tcell-mediated immune response in the vaccinated mice. Two
weeks after boosting, T-cell proliferation was examined in
splenocytes from VV-immunized mice. Immunization with VV
expressing the chimeric protein (VV-GM–gp120) significantly
increased the T-cell response to Env protein compared with
that of VV-ENV-immunized mice, with SI of 13n7 and 6n1,
respectively (Fig. 4a). Control mice immunized with VV
expressing GM-CSF did not induce T-cell proliferative responses after stimulation with soluble gp120. ELISPOT assays
were performed with splenocytes from VV recombinantimmunized mice to study further the enhancement of cellular
activity. Spleen cells from mice immunized with VV-GM–
gp120 showed a twofold increase in the number of IFN-γsecreting cells compared with those from mice immunized with
VV-ENV (68\10& versus 36\10&) ; VV-GM-CSF-immunized
mice showed only 2\10& (Fig. 4 b).
These results indicate that the GM-CSF portion of the
chimeric protein potentiates the VV recombinant-induced Tcell response to HIV-1 Env (Fig. 4).
Discussion
VV recombinants expressing HIV-1 envelope antigens
were one of the first prototype vaccine constructs generated
against AIDS (Chakrabarty et al., 1986 ; Hu et al., 1986). In
animal models, these constructs elicit both humoral and cellular
immune responses (Earl et al., 1989). Virus-specific cytotoxic T
lymphocytes (CTL) are generally believed to be the best
candidates for control of virus replication. The dramatic
decrease in HIV-1 virus load following the initial appearance of
CTL after primary infection and the temporal association
between HIV-1-specific CTL activity and stable virus load or
14
Stimulation index
12
(a)
10
8
6
4
2
0
70
IFN-γ secreting cells/10 5
spleen cells
recombinants. Anti-gp120 reactivity in pooled serum was
analysed by ELISA in 96-well gp120-coated plates. Both VVENV and VV-GM–gp120 vaccination elicited similar levels of
anti-HIV-1 Env antibodies (Fig. 3a) ; the anti-gp120 antibody
titre in VV-ENV-immunized mice was 3n7i10%, while that for
VV-GM–gp120 was 3n2i10%.
To characterize the response further, sera from both groups
were tested against a panel of overlapping peptides encompassing the entire gp120 protein. Different recognition patterns
were observed ; while sera from VV-ENV-immunized mice
reacted only with peptides of the third hypervariable loop (V3)
(peptides 27–28 corresponding to amino acids 262–290),
antibodies induced by the VV-GM–gp120 immunogen recognized not only the V3 loop but also other regions, specifically
the gp120 C-terminal domain (peptides 46 and 47, amino acids
452–479) (Fig. 3 b).
60
VV-GM-CSF
VV-GM – gp120
VV-ENV
VV-GM – gp120
VV-ENV
(b)
50
40
30
20
10
0
VV-GM-CSF
Fig. 4. Cellular anti-HIV-1 gp120 immune response induced after
vaccination of mice with VV-Env recombinants. (a) Proliferative response
of spleen cells from mice primed and boosted with VV recombinants as
indicated in the legend to Fig. 3. Spleens were removed 14 days after
boosting and spleen cells were incubated with (filled bars) or without
(hatched bars) purified gp120 (1 µg/ml). [3H]Thymidine incorporation
was measured and the SI was calculated as described in Methods. (b) V3
loop-specific CD8+ T-cell response in spleen cells from mice immunized
as described above. The number of IFN-γ-secreting CD8+ cells was
quantified by the ELISPOT assay, following the protocol described in
Methods, after stimulation of the splenocytes with V3 loop peptidepresenting APC.
CD4+ cell counts during asymptomatic stages are the best
indicators of CTL efficiency. A good correlation has also
recently been reported between long-term survival and CTL
activity in paediatric HIV-1-infected patients (Luzuriaga &
Sullivan, 1993). The mechanisms involved in this CTL
protection include the elimination of virus-infected cells and
production of suppressive factors, creating a balance between
virus elimination and replication.
We have studied the potential enhancement of immune
responses to HIV-1 gp120 in mice immunized with a chimeric
GM-CSF–gp120 protein expressed by a recombinant VV.
Immunization with antigens fused to GM-CSF facilitates
primary immune responses (Tao & Levy, 1993) and, by using
two previously characterized HIV-1 MN vaccine constructs,
Ahlers et al. (1997) showed that GM-CSF is the most effective
cytokine for enhancing cellular and humoral immunity. Here,
we generated VV recombinants expressing murine GM-CSF
(VV-GM-CSF), the HIV-1 Env protein (VV-ENV) or a chimeric
antigen consisting of the HIV-1 Env protein fused to GM-CSF
(VV-GM–gp120). Western blot analysis of VV-GM–gp120infected cells showed a product of approximately 150 kDa that
was recognized by different gp120-specific antibodies. The
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D. Rodrı! guez and others
fusion protein was efficiently released to the medium from cells
and was biologically active, since culture supernatants from
infected cells promoted proliferation of a GM-CSF-dependent
cell line. This activity was somewhat lower, however, compared with the activity of native GM-CSF expressed by VVGM-CSF. The gp120 moiety retained its ability to bind
specifically to the human CD4 receptor.
BALB\c mice immunized with VV-GM–gp120 developed
a broader spectrum of anti-gp120 antibodies than animals
immunized with a VV recombinant expressing the non-fused
Env antigen (VV-ENV). Epitopes corresponding to the V3
loop of the envelope protein were recognized equally well by
serum from both groups of mice. In addition, VV-GM–gp120induced antibodies recognized peptides corresponding to the
C terminus of gp120 that were not recognized by antibodies
elicited after VV-ENV immunization. These results could be
explained by differential folding and presentation of the HIV
Env protein to the immune system when fused to GM-CSF.
Although the V3 loop is the principal neutralizing linear
determinant in the Env protein, the sequence variability of this
segment gives rise to limited antibody specificity, reducing the
potential utility of a protective antibody response (Goudsmit et
al., 1991). This problem has intensified interest in neutralization
sites outside the V3 loop. Neutralizing epitopes have been
reported in the gp120 C terminus, in a highly immunogenic
region of gp41 and in the C terminus of gp41 (Ho et al., 1987).
We also observed enhancement of the anti-gp120 cellular
immune response in animals immunized with VV expressing
the chimeric GM–gp120 antigen. Both the T-cell proliferative
responses and the number of IFN-γ-secreting cells, as determined in the ELISPOT assay, showed an approximately
twofold increase in splenocytes from animals immunized with
the fusion protein when compared with those of animals
immunized with Env alone. The results of the ELISPOT assay
specifically indicate an increase in the anti-gp120 CD8+ T-cell
response induced by the recombinant virus expressing the
chimeric GM–gp120 antigen.
These results show that GM-CSF contributes to the
enhancement of the cellular immune response against the HIV1 Env antigen elicited by recombinant VV and that coexpression of this immunomodulatory cytokine may represent
a means of improving the efficiency of VV-based anti-HIV-1
vaccines.
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for technical assistance. The Department of Immunology and Oncology
was founded and is supported by the CSIC and Pharmacia & Upjohn. This
work was supported in part by a grant from CICYT of Spain SAF95-0072
(to M. E.). D. R. and J. R. R. were recipients of contracts from the CSIC of
Spain.
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