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Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma, Madrid, Spain; and
Department of Medical and Molecular Parasitology, New York University Medical Center, New York, NY 10010
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in serum and spleen early after
infection. Enzyme-linked immunospot analysis of mice inoculated with
rVVlucIL-12, revealed a nearly 2-fold increase in the number of
specific anti-VV CD8+ T cells compared with that in
mice given control rVV, and the serum Ab response was biased in favor
of a Th1 response. An enhancement of about 2-fold in the number of
anti-gp160 IFN--secreting CD8+ T cells was observed
in mice inoculated with rVVenvIL-12, when a dose of 1 x
107 PFU/mouse was used, but this enhancement was not
observed when mice were given 5 x 107 PFU. This
variation with virus dosage was confirmed in mice immunized
simultaneously with different multiplicities of rVV expressing singly
the env or IL-12 genes. The highest specific CMI was
obtained in mice coadministered a low dose (2 x 104
PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our
findings provide evidence for specific enhancement of the CMI to HIV-1
Env by the differential expression of IL-12 and env
genes delivered from VV recombinants. This approach can be of wide
vaccination interest as a means to improve immune responses to other
Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The understanding of the immune response generated during HIV-1 infection has revealed that neutralizing Abs arise, but may not be critical in limiting viral replication due to genetic variability of the virus (6). In addition, different vaccination approaches in simian animal models or in human trials failed to elicit protective neutralizing Abs (7). On the contrary, several studies have emphasized the importance of CTL in combating HIV-1 infection and controlling the development of AIDS. CTL activity coincides with the early containment of HIV-1 replication (7) and correlates with stable clinical status and low virus load in chronically infected individuals (8, 9, 10). Moreover, high anti-HIV CTL responses are frequently observed in healthy women repeatedly exposed to the virus during unprotective sexual practices (11) and have also been demonstrated in uninfected infants born to HIV-1-positive mothers (12). In the SIV infection model, protection after vaccination has been correlated with a strong CTL response over other immunological parameters (13). Consequently, of a spectrum of various host immune responses, induction of cell-mediated immunity (CMI) might be an important requirement in an effective candidate HIV-1 vaccine.
The development of an effective CMI response after vaccination rests on
an extensive scope of factors, among which cytokines present during the
priming could play a critical role. Two different subsets of
CD4+ T lymphocytes, Th1 and Th2, differing in the pattern
of cytokines produced, have been described to be crucial in the
generation of a cellular or an Ab immune response, respectively.
Different lines of evidence showed that the early decision toward Th1-
or Th2-type immune response is mainly dependent on the balance between
IL-12 (which favors a Th1 response) and IL-4 (which favors a Th2
response). Among the main functional features of IL-12 are 1) it
potentiates cytokine production, particularly IFN-, in T lymphocytes
and NK cells; 2) it acts as a growth factor for preactivated T and NK
cells; and 3) it is involved in the generation of CTLs and in the
activation of cytotoxicity in both CD8+ T and NK cells. In
addition, IL-12 has a prominent role in the generation of Th1 cells and
the optimal differentiation of CTLs (14). Thus, to induce strong and
stable CMI responses against HIV-1 infection, the use of vectors
delivering cytokines capable of triggering a Th1 response in
conjunction with appropriate Ags is a encouraging approach. In this
regard, different vaccination strategies with IL-12 delivered as a
soluble product, expressed from DNA vectors or viral vectors, have
provided evidence for enhancement of CTL responses that correlated with
regression of tumors (15), resolution of autoimmune diseases (16), and
protection against various intracellular pathogens (17) and against the
development of murine AIDS (18).
In addition to the promising prophylactic and therapeutic advantages of the use of live vectors delivering immunomodulators, the potential capability of the encoded cytokine to modulate the vector pathogenicity might be a desirable property, especially in the case of live virus-based vaccines by decreasing the risk of side-effects during virus infections. Indeed, different recombinant virus-encoding cytokines have been described as immunological tools for dissecting the in vivo functions of cytokines in antiviral immunity. Expression of cytokines from rVV have been reported to have a profound effect on viral infection (19), but their induction and their involvement in promoting specific immune responses to Ags are poorly characterized events. Hence, analysis of the effect of cytokines on the virus vector itself should be explored to understand the implications for the Ag-specific immune responses elicited when both Ag and cytokine are delivered from a live-based vector.
In this investigation we have defined the antiviral and immunological
roles of IL-12 when expressed from VV in the absence and the presence
of HIV-1 Env. Our findings demonstrate that rVV expressing IL-12 genes
are safe vectors, since VV replication is severely compromised through
the induction of IFN-. In immunized mice IL-12 expression drives a
Th1-type response against both the vector and the env gene
product, leading to an enhanced cellular immune response and a biased
serum Ab response in favor of IgG2a subclass. Moreover, we show that
the dosage of the rVV expressing IL-12 and env genes plays
an essential role in the enhancement of the cellular immune response
against the HIV-1 gp160 protein, and that by coexpressing IL-12 and
env genes in different ratios it is possible to trigger a
desirable cellular immune response to the HIV-1 Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The VV recombinants employed in this study derived from the wild-type WR strain, rVVluc (expressing the luciferase and ß-galactosidase genes) and rVVenv (expressing the entire env gene of HIV-1 strain IIIB and ß-galactosidase gene), have been described previously (20, 21). The recombinant viruses rVVlucIL-12 (expressing the luciferase gene and p35 and p40 IL-12 subunits), its control rVVlucHA- (expressing the luciferase gene and insertional inactivated in the HA gene), as well as rVVenvIL-12 and rVVenvHA- were generated for this study, and their construction is described below. Viruses were grown in HeLa cells, titrated in African green monkey kidney BSC-40 cells, and purified as described previously (22).
Engineering of the VV recombinant viruses
The cDNAs coding for both IL-12 subunits (p35 and p40) linked by
an internal ribosomal entry site sequence (IRES) were isolated from
plasmid pBS-IL-12 by digestion with the restriction enzymes
EcoRI and BamHI. The DNA fragment containing the
complete IL-12 sequence (p35-IRES-p40) was blunt ended by treatment
with the large fragment of the Escherichia coli DNA
polymerase I (Klenow) and cloned into the SmaI site of the
VV insertion vector pJR101. As a result of this cloning strategy, we
isolated a plasmid, pJR101-IL-12, that contains the IL-12 (p35-IRES-p40
cassette) genes under the control of a VV synthetic early/late promoter
e/l (23), the E. coli ß-glucuronidase marker gene under
the control of the VV early/late promoter p7.5, and all these sequences
flanked by regions from VV hemagglutinin (HA) gene. Double rVV were
prepared by infecting BSC-40 cells with either the VVenv or the VVluc
recombinant virus and transfecting them with the plasmid pJR101-IL-12.
Cell cultures were harvested at 48 h postinoculation (hpi), and
the double-recombinant viruses were selected after plaque assay by the
addition of X-Gluc to the agar overlay (24). By this procedure, rVV
containing the HIV-1 env gene (VVenvIL-12) or the
luciferase gene (VVlucIL-12) into the TK region and the IL-12 cassette
into the HA locus were isolated. After three rounds of selection,
viruses were purified following standard procedures. A similar
strategy was followed to generate control viruses VVlucHA- or
VVenvHA-, but in this case, transfection was performed with
empty VV insertion plasmid pJR101. In Fig. 1
A is shown a schematic
representation of the different rVV constructed for this study. In Fig. 1B is shown IL-12 expression in extracts from rVV
BSC-40-infected cells by Western blot analysis. An IL-12 bioassay was
also performed with the same samples, indicating that IL-12 expressed
from rVV was bioactive (data not shown).
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BALB/c mice (H-2d; 6–8 wk old) were immunized i.p. with different doses (indicated as PFU) of the different rVV in 200 µl of sterile PBS. Fourteen days after virus inoculation, blood was obtained from the retro-orbital plexus by a heparinized capillary tube, collected in an Eppendorf tube, and centrifuged, and serum was obtained and stored at -20°C.
Measurement of luciferase activity in mice tissues
Replication of rVV in different mouse tissues was followed by a highly sensitive luciferase assay, previously described (20). Different groups of mice received an i.p. inoculation with 5 x 107 PFU/mouse of the recombinant viruses: rVVluc, rVVlucHA-, or rVVlucIL-12. At various times postinoculation animals were sacrificed, and spleens, livers, and ovaries were resected, washed with sterile PBS, weighed, and stored at -70°C. Then, tissues from individual mice were homogenized in luciferase extraction buffer (300 µl/spleen extract and 100 µl/ovary extract) containing 1% Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 1 mM PMSF, 100 µg/ml soybean trypsin inhibitor, and 10 µg/ml leupeptin. The luciferase activity was measured in the presence of luciferin and ATP using a Lumat LB 9501 Berthold luminometer (Berthold, Nashua, NH), and it was expressed as relative luciferase units per milligram of protein. Protein content in tissue extracts was measured employing the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL).
RNA extraction
Total mRNA was purified from aseptically removed spleens. Relatively identical small pieces of spleens from three mice per group were pooled and homogenized in extraction buffer using an Ultraturrax T8 mechanical homogenizer (Janke & Kunkel, Staufen, Germany). Clear lysates were source for mRNA purification using QuickPrep Micro mRNA purification kit (Pharmacia, Uppsala, Sweden) following the instructions of the manufacturer.
Amplification of mRNA by RT-PCR
Semiquantitative RT-PCR was performed on mRNA using the
SuperScript One-Step RT-PCR System (Life Technologies, Gaithersburg,
MD). The RT conditions used were 30 min at 50°C followed by a
denaturing step at 94°C for 2 min, followed by 30 (for hypoxanthine
phosphoribosyltransferase, HPRT) or 40 (for IFN- and IL-12) cycles.
The number of cycles was adjusted for every pair of primers to get a
linear range during the reactions. Cycling conditions were 94°C for
30 s, 60°C (58°C for IL-12) for 30 s, and 68°C for 1.5
min, followed by a final extension step at 68°C for 5 min. Primers
sequences were: for HPRT, 5'-GTTGGATACAGGCCAGACTTTGTTG-3' (sense)
and 5'-GATTCAACTTGCGCTCATCTTAGGC-3' (antisense); for IFN-,
5'-TGAACGCTACACACTGCATCTTGG-3' (sense) and
5'-CGACTCCTTTTCCGCTTCCTGAG-3' (antisense); and for IL-12 p40
subunit, 5'-CTCACATCTGCTGCTCCACAA-3' (sense) and
5'-CTCCTTCATCTTTTCTTTCTT-3' (antisense). PCR products were analyzed by
ethidium bromide staining after electrophoresis on 1.2% agarose gels.
Ab measurements by ELISA
ELISA was used to determine the presence of Abs against VV Ags in serum samples. The VV Ags employed to coat 96-well flat-bottom plates at a concentration of 1 µg/ml consisted of envelope proteins extracted from purified virions, as described previously (25). VV Ags were suspended in carbonate buffer, pH 9.6, plated at 50 µl/well, and incubated overnight at 4°C. Afterward, the contents of the wells were discarded and washed three times with PBS plus 0.05% Tween-20 (PBS-T), and blocking buffer (borate-buffered saline with 1% BSA, 1 mM EDTA, and 0.05% Tween-20) was added at 100 µl/well and incubated for 1 h at 37°C. The plates were washed once with PBS-T, and samples diluted in blocking buffer were added in a volume of 100 µl/well and incubated for 1 h at 37°C. Then, plates were washed three times before the detection Ab was added. Peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Southern Biotechnology Associates, Birmingham, AL) Abs were diluted 1/1000, 1/1500, and 1/2000 respectively in blocking buffer and incubated for 1 h at 37°C. After washing the plates three times with PBS-T, the wells were reacted with the peroxidase substrate O-phenylendiamine dihydrochloride (Sigma, St. Louis, MO). After 10–15 min of incubation at room temperature, the reaction was stopped by adding 2 N H2SO4, and the absorbance values were measured at 492 nm on a Labsystems Multiskan Plus plate reader (Chicago, IL).
T cell proliferation assays
Lymphocytes were removed from spleens by passing tissues through
a sterile mesh to obtain cell suspensions. Cells were suspended in
complete medium (RPMI 1640 supplemented with 10% FCS, 2 mM
L-glutamine, and 10 mM 2-ME). RBC in preparations of spleen
cells were lysed with 0.1 M ammonium chloride buffer. Splenocytes were
cultured in triplicate (106 cells/well) in 96-well
microtiter flat-bottom plates and stimulated with purified VV
previously inactivated by UV light at 1 µg/ml, purified gp160 protein
(Intracel, Cambridge, MA; 1 µg/ml), or Con A (1 µg/ml; Sigma).
Plates were incubated for 3 days at 37°C in 5% CO2.
After this incubation period, proliferation assays were conducted by
labeling the cells with [3H]thymidine (1 µCi/well) for
18 h. Following automated harvesting, [3H]thymidine
incorporation was measured by liquid scintillation counting. Cytokine
levels in culture supernatants were determined after 48 h (IL-10)
or 72 h (IFN-) of incubation. Supernatants from triplicate
cultures were pooled and stored at -70°C until performing the assay.
Evaluation of cytokines levels by ELISA
Cytokine levels in culture supernatants and sera were determined by ELISA using the appropriate combination of Abs from Genzyme (Cambridge, MA). Briefly, 96-well flat-bottom plates were coated with 100 µl of anti-cytokine Abs diluted in the buffer specified by the manufacturer and incubated overnight at 4°C. The wells were then washed with PBS-T and coated with PBS containing 1% BSA at 37°C for 2 h. Serial 2-fold dilutions of supernatants or sera and adequate dilutions of standard cytokines were added in duplicate and incubated at 37°C for 1–2 h. The wells were then washed with PBS-T and incubated with the specific biotinylated anti-cytokine Ab diluted in PBS-T with 1% BSA for 1–2 h. After three or four washes, wells were incubated with HRP-conjugated streptavidin for 15 min at 37°C and developed with TMB reagent (Sigma). The reaction stopped with 2 N SO4H2, and the absorbance values were measured at 450 nm.
Evaluation of CD8+ T cells by the ELISPOT assay
The ELISPOT assay to detect Ag-specific CD8+ T cells
was performed as previously described (26). Briefly, 96-well
nitrocellulose plates were coated with 8 µg/ml of anti-mouse
IFN- mAb R4–6A2 (PharMingen, San Diego, CA) in 75 µl of PBS.
After overnight incubation at room temperature, wells were washed three
times with RPMI 1640, and 100 µl of complete medium supplemented with
10% FCS was added to each well. Afterward, the plate was incubated at
37°C for at 1 h. Spleen cells (depleted of RBC) from different
groups of mice were added in triplicate at 2-fold dilutions. P815 cells
(a mastocytome cell line that expresses only MHC class I molecules)
were used as APC. When the number of specific CD8+ T cell
anti-VV Ags was evaluated, P815 cells (107 cells/ml)
were infected at a multiplicity of infection of 5 PFU/cell, and at 4.5
hpi, cells were washed and treated with mitomycin C (30 µg/ml;
Sigma). When the number of CD8+ IFN--secreting cells
specific for the V3 loop epitope of the HIV-1 Env protein was
evaluated, P815 cells were pulsed with 10-6 M of the
synthetic peptide GPGRATVTI (9 Env) or RGPGRAFVTI (10 Env) and treated
with mitomycin C as described above. After several washes with culture
medium, 105 P815 cells were added to each well. As a
control, P815 cells not pulsed with the peptide or uninfected but
treated under similar conditions were used. Plates were incubated for
24 h in a 37°C incubator with a 5% CO2 atmosphere,
washed extensively with PBS-T, and incubated overnight at 4°C with a
solution of 2 µg/ml of biotinylated anti-mouse IFN- mAb XMG1.2
(PharMingen) in PBS-T. Thereafter, plates were washed with PBS-T, and
100 µl of peroxidase-labeled avidin (Sigma) at a 1/800 dilution in
PBS-T was added to each well and incubated at room temperature. One
hour later, wells were washed with PBS-T and PBS. The spots were
developed by adding 1 µg/ml of the substrate 3,3'-diaminobenzidine
tetrahydrochloride (Sigma) in 50 mM Tris-HCl, pH 7.5, containing
0,015% hydrogen peroxide. Then spots were counted with the aid of a
stereomicroscope.
IL-12 bioassay
Biologically active IL-12 was measured in supernatants from rVV
expressing IL-12 BSC-40-infected cells and in serum samples from
inoculated mice. The indicator WEHI 279 cells (European Cell Culture
Collection, Salisbury, U.K.), a mouse B cell lymphoma, was used to
titrate the bioactive IL-12 as previously described (27). Briefly,
flat-bottom 96-well dishes were coated with a rat mAb against mouse
IL-12 (nonneutralizing C15 rat mAb, Genzyme) and incubated overnight at
4°C. Afterward, plates were washed and blocked with filtered PBS-T
with 1% BSA for 1 h at 37°C. Dilutions of samples and standard
recombinant mouse IL-12 (Genzyme) were added and stored at 37°C for
4 h. Freshly prepared splenocytes were added at 106
cells/well and cultured for 48 h in the presence of 50 U/ml of
rIL-2. Culture supernatants were mixed with 104 cells/well
of exponentially growing WEHI 279 cells and cultured for an additional
72 h. IFN- inhibition of indicator cells growth was measured by
the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
colorimetric assay. Absorbance values were introduced in the standard
curve and transformed to IL-12 units according to the manufacturer’s
data (8000 U/µg).
CD8+ T cell purification
CD8+ cells were purified using MACS High Gradient Magnetic Separation Columns VS+ (Miltenyi Biotec, Bergisch Gladbach, Germany) for positive selection from whole splenic populations following the manufacturer’s procedure. Cells were labeled with CD8a (Ly2.53–6.7) Micro Beads (Miltenyi Biotec), and 108 cells/ml were loaded in the column. A fraction of the whole population or positive and negative eluates were formaldehyde fixed and labeled with fluorescence-specific anti-CD4 and anti-CD8 Abs for cell sorting by flow cytometry (FACS, Becton Dickinson, Mountain View, CA). The sorted profiles were used to evaluate the accuracy of the purification and quantitate the ELISPOT assay performed with those populations. Less than 3% of CD8+ cells were present in the CD8+-depleted fraction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little information is available on the effect of IL-12 on VV
infection; hence, our first goal was to evaluate the effect of IL-12 on
the capability of the viral vector to infect and persist in target
tissues. Thus, we studied the replication of a WR-based rVV that
expresses the IL-12 gene (rVVlucIL-12) by measuring the activity of the
coexpressed luciferase reporter gene. As the IL-12 expression cassette
was inserted into the HA gene, and as it is known that inactivation of
this gene reduces VV infectivity in vivo (28), we employed as controls
two luciferase expression viruses rVVlucHA- and
rVVlucHA+ with an inactivated or an intact HA gene,
respectively. To evaluate in vitro whether IL-12 expression could have
any effect on VV replication, BSC40 cells were infected (10 PFU/cell)
with the three different VV recombinants (rVVlucIL-12,
rVVlucHA-, and rVVluc) and at different times postinfection
(5, 18, and 24 hpi) luciferase activity was measured in cell extracts.
No significant differences in luciferase activity were observed among
the different rVV (data not shown), indicating that IL-12 expression
had no effect on viral replication. To evaluate in vivo the effect of
IL-12 expression on VV replication, groups of mice were i.p. inoculated
with a single dose of 5 x 107 PFU/mice of rVVluc,
rVVlucHA-, or rVVlucIL-12, and three animals per day and
group were sacrificed on days 1, 2, 3, 4, and 7 postinoculation (dpi).
Luciferase activity in spleen and ovaries of individual samples were
measured, and results are depicted in Fig. 2. At 1 dpi, similar luciferase values
were detected in spleen samples from mice inoculated with either
rVVlucIL-12 or rVVlucHA-, and these were about 50-fold
lower than those found after infection with rVVluc. However, at 2 dpi
luciferase levels fell 100-fold in animals given the IL-12-expressing
virus, and no infectious virus was detected by plaque assay in this
group (not shown). By this time, levels of replication of
rVVlucHA- or rVVluc control viruses remained essentially
stable. At 3 dpi only background luciferase activity was measured in
all mice given the rVVlucIL-12, whereas low, but still measurable,
luciferase activity was detected in mice inoculated with control
viruses. The kinetics of the rVVlucHA- virus
were parallel to those of control rVVluc virus, but infection in the
spleen was resolved later in animals inoculated with rVVluc virus.
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multiplication of rVV in ovaries
was extended for the three viruses analyzed compared with that in the
spleen. In this organ, differences between rVVlucHA- and
rVVlucIL-12 were more pronounced than those in spleen. At 1 dpi,
luciferase activity in ovaries was 50-fold lower in animals inoculated
with rVV expressing IL-12 than in animals inoculated with the control
virus (rVVlucHA-). By 2 dpi, luciferase activity reached a
peak in both rVVlucHA-- and rVVluc-inoculated groups,
whereas 100-fold lower activity was detected in mice infected with
rVVlucIL-12. In all groups, luciferase expression in ovaries declined
by day 3 and was greatly reduced by 4 dpi in animals receiving IL-12. A
slower decrease in luciferase levels was observed in mice given control
viruses, especially in those receiving rVVluc.
The results shown in Fig. 2 demonstrate that IL-12 expression during VV
infection impairs virus multiplication and promotes a rapid clearance
of the virus from infected tissues, suggesting that delivery of IL-12
ensures attenuation of VV along the infectious process in mice.
Induction of IL-12 and IFN- in vivo after inoculation of mice
with a rVV expressing IL-12
To determine whether the decreased infectivity observed for
rVVlucIL-12 in mice could be correlated with induction of
IFN- expression, we next analyzed the levels of IL-12 and IFN-
in serum samples from mice inoculated with rVVlucIL-12 or
rVVlucHA-. Due to the rapid clearance observed
for VV after IL-12 expression (Fig. 2), we examined the levels of these
cytokines at early times postinfection. Thus, serum samples were
collected every 6 h during the first dpi and daily
thereafter until 7 dpi. IL-12 was measured both by ELISA against the
p40 subunit (data not shown) and by determination of bioactive p70
heterodimeric form (Fig. 3A, upper
panel), rendering comparable results. The maximum amount of IL-12
was found at 6 hpi in the inoculated groups. Nevertheless, at least
5-fold higher levels were found in samples from rVVlucIL-12-immunized
mice than in animals inoculated with the control virus. Levels of IL-12
in the control group fell into the detection limit of the assay (<0.64
U/ml) beyond 12 hpi and were the same as those found in naive mice (not
shown). The higher levels of IL-12 (ranging from 480–240 U/ml) were
found during the first dpi in samples from mice inoculated with
rVVlucIL-12, but IL-12 was still detected at 3 and 4 dpi. This result
clearly demonstrates that there is a rapid induction of IL-12 upon
infection with rVV expressing this cytokine, and significant levels are
still present during the clearance period of the virus (see Fig. 2).
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, evaluated by ELISA, were detected in serum from
mice inoculated with IL-12 expressing rVV. The maximum level of IFN-
appeared, with a lag of 12 h with respect to IL-12 production,
around 18 hpi in rVVlucIL-12-immunized mice. High levels were still
present at 2 dpi, falling to background levels by 4 dpi, while in the
control group detectable levels were only found at 6 and 12 hpi (Fig. 3A, lower panel).
The kinetics of mRNA expression for the inducible p40 subunit of IL-12
and for IFN- in the spleen were analyzed by semiquantitative RT-PCR
(Fig. 3B). Induction of IL-12 mRNA was observed at 6 hpi in
mice inoculated with rVVlucIL-12, increased about 10 times by 18 hpi
and declined by 1 dpi, whereas in mice inoculated with the control
rVVlucHA- virus, low but consistent amounts of p40 mRNA
were present at 18 hpi. Levels of IFN- mRNA in the spleens of mice
inoculated with the rVVluc-IL12 peaked at 24 hpi and were detectable
until 2 dpi (Fig. 3B and data not shown). The induction of
IFN- mRNA was clearly detected at 18 hpi in mice inoculated with the
control rVV, and the levels were >10 times lower than those in mice
inoculated with rVVlucIL-12. Under the conditions of the assay, spleens
from naive noninoculated mice did not reveal detectable mRNAs for the
two cytokines.
Evaluation of the anti-VV immune response elicited in mice inoculated with rVVlucIL-12
The findings presented in Figs. 2 and 3 clearly reveal that IL-12
has a profound effect on the replication of VV and that this cytokine
potentiates IFN- production. As both cytokines could have a major
impact on the modulation of host immune responses, our next approach
was to analyze the role of IL-12 in specific immune responses against
the VV vector.
CD8+ IFN--secreting T cells against VV
As IL-12 has the capacity to augment cell-mediated immune
responses (30, 31), and as CD8+ CTL responses are involved
in the resolution of infection by poxviruses (32, 33), we first
evaluated the CD8+ T cell immune response elicited against
VV following expression of IL-12 in mice inoculated with rVVlucIL-12.
We employed a modification (34) of the ELISPOT assay that quantifies
the number of specific anti-VV MHC class I-restricted
IFN--secreting cells. Groups of three or four mice were i.p.
inoculated with 5 x 107 PFU/mice of rVVlucIL-12 or
rVVluc-HA-, and at 7 or 14 dpi the number of anti-VV
IFN--secreting CD8+ T cells was determined. As shown in
Fig. 4A, at 7 dpi the numbers
of specific IFN--secreting CD8+ T cells in spleens from
animals inoculated with rVVlucIL-12 (1641 ± 104/106
cells) were significantly different (p < 0.05)
with respect to those in mice inoculated with the control virus
(964 ± 64/106 cells). This represents 1.7-fold more
MHC I class-restricted anti-VV IFN--secreting cells in animals
inoculated with rVVlucIL-12 virus than in the control group. As
expected, by 14 dpi the number of specific anti-VV CD8+
T cell IFN--secreting cells decreased in the two groups of animals
(Fig. 4A), but differences between the groups were
maintained. The number of specific CD8+ T cells quantified
after purification revealed no differences when the total population
was compared with the CD8+-selected fraction (Fig. 4B). These observations confirm that in the ELISPOT assay
shown in Fig. 4A the MHC class I-restricted cell population
responsible for IFN- secretion was mainly CD8+ T cells.
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We next investigated the influence of IL-12 expressed from VV on
the pattern of cytokines expressed by T cells in vitro after Ag
restimulation. Two groups of mice were immunized i.p. with 5 x
107 PFU of rVVlucIL-12 or control rVVlucHA-,
and 14 days later splenocytes from both groups of mice were
restimulated in vitro with UV-inactivated VV. As shown in Fig. 4C, high levels of IFN- (Th1-type cytokine) were found in
splenocyte cultures restimulated with VV in both groups of animals,
with a slight increase in rVVlucIL-12 with respect to control virus.
However, levels of IL-10 (Th2-type cytokine) were significantly
decreased in supernatants of splenocytes from rVVlucIL-12 compared with
controls, suggesting that IL-12 was suppressing an antiviral Th2 type
of response, rather than enhancing the CD4+ Th1 response.
Effect of IL-12 delivered by rVV on systemic Ab response to VV Ags
In murine systems it has been shown that Th2 cytokines favor the
induction of IgG1 subclass Abs, whereas IgG2a subclass Abs are induced
in a context of Th1 cytokines (35). Thus, we next evaluated anti-VV
IgG subclasses in sera, 2 wk after inoculation of mice with rVVlucIL-12
or rVVlucHA-. As shown in Fig. 5, there were no major differences in
levels of specific IgG or IgG2a Abs between the groups, while in
rVVlucIL-12-inoculated mice, anti-VV IgG1 subclass Abs were greatly
reduced compared with the levels found in control mice. Thus, the ratio
of IgG2a/IgG1 Abs (Fig. 5, right panel) in mice inoculated
with rVVlucHA- was 1.7, and this ratio was increased 13.5 times in sera
from mice inoculated with rVVlucIL-12. These findings indicate that
IL-12 expressed from rVV modulates the humoral immune response by
down-regulating specific IgG1 subclass Ab (Th2 cytokine) production,
rather than by promoting up-regulation of the IgG2a subclass (Th1
cytokine), a process resembling in vitro restimulation assays (see Fig. 4C).
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To assess whether IL-12 delivered by VV might be effective in
potentiating the cellular immune response against the Env protein of
HIV-1, we first evaluated the IL-12 action when the Ag and the cytokine
were coexpressed from the same virus vector. To this aim, groups of
mice were immunized i.p. with either 5 x 107 or
1 x 107 PFU/mouse of rVVenvHA- (that expresses the
complete env of HIV-1 strain IIIB) or rVVenvIL-12
(coexpressing env and IL-12 genes). Fourteen days after
immunization, we evaluated the numbers of specific splenic
IFN--secreting CD8+ T cells, using P815 cells pulsed
with a CD8+ T cell peptide specific for the V3 loop of
env. Immunization with 1 x 107 PFU of
rVVenvIL-12 induced about 2-fold higher number of splenic Env-specific
IFN--secreting CD8+ T cells with respect to spleen cells
from mice inoculated with control virus (p <
0.01; Fig. 6A). However, this
immune response was 3-fold lower than that in control when mice were
inoculated with the higher dose (5 x 107 PFU) of rVVs
(Fig. 5A). Indeed, levels of IFN- and IL-10 measured 14
dpi after Ag in vitro restimulation of splenocytes from mice inoculated
with 1 x 107 PFU of rVVenvIL-12 were 3-fold higher
and 10-fold lower, respectively, than those found in control samples,
indicating that an increase in the Th1-type immune response was
occurring at the low dose (1 x 107 PFU) of infection
with the IL-12-expressing rVV (data not shown).
|
, and the extent of viral clearance during
short times after rVV inoculation. To this aim, groups of four mice
were inoculated i.p. with either 5 x 107 or 1 x
107 PFU/mouse of rVVlucHA- or rVVlucIL-12, which as live
vectors are phenotypically undistinguishable from rVV expressing Env,
but allowed measurement of VV replication following luciferase activity
in tissues. Levels of IL-12 in serum at 12 hpi were 10- or 50-fold
higher in mice inoculated with 1 x 107 or 5 x
107 PFU of rVVlucIL-12, respectively, than in the
corresponding control groups (Fig. 6B). However, levels of
IFN- induced were 10-fold higher in mice inoculated with 5 x
107 PFU of rVVlucIL-12 than in control immunized mice, but
lower levels of IFN- (20–120 pg/ml) were present in mice inoculated
with 1 x 107 PFU of either control or
IL-12-expressing rVV viruses. Interestingly, by the second dpi 50-fold
lower luciferase activity was found in mice inoculated with 5 x
107 PFU of rVVlucIL-12 compared with that in control
infected mice (Fig. 2), while at this time luciferase levels were
identical in mice given 1 x 107 PFU of either
rVVlucHA- or rVVlucIL-12 (data not shown).
The results presented in Fig. 6 showed that IL-12 levels can be
controlled by the dose of rVV expressing IL-12 inoculated, which seems
to be critical for the extent of cellular immune responses to Env.
Enhancement of the immune response to HIV-1 Env by delivering Env and IL-12 from two different rVV vectors
To more accurately explore the dose-dependent action of IL-12 on
HIV-1 Env, we tried to modulate the immune response by delivering Env
and IL-12 from two different vectors. Groups of four mice were
inoculated i.p. with 1 x 107 PFU/mouse of rVVenvHA-
alone or in combination with different doses (2 x
104, 2 x 105, and 2 x
106 PFU) of rVVlucIL-12 (as a source of IL-12), and 14 days
later the number of specific splenic CTLs was determined by ELISPOT
(Fig. 7A). The results
obtained revealed that coadministration of 2 x 104
PFU of the rVV expressing IL-12 with 1 x 107 PFU of
rVVenvHA- increased by 3 times (p < 0.001) the
number of specific IFN--secreting CD8+ T cells. In
contrast, groups of mice inoculated with higher doses of the rVV
expressing IL-12 (2 x 105 and 2 x
106 PFU) showed no significant differences
(p > 0.2) with respect to the control
group inoculated only with rVVenvHA-. To further investigate the IL-12
enhancement of cellular activity we performed Th cell proliferation
assays 2 wk after immunization with splenocytes from immunized mice.
Fig. 7B (left panel) shows similar
stimulation indexes in spleen cells from mice immunized with rVVenv
alone (1 x 107 PFU) or with the same dose of rVV env
and 2 x 106 PFU of rVVlucIL-12. However, a nearly
3-fold increase in specific T cell proliferation activity appeared in
the mice receiving the lower dose of rVVlucIL-12 virus (2 x
104 PFU). Since IFN- production by CD4+ T
cells is the most reliable indicator of a Th1 phenotype, we also
measured the levels of IFN- secreted in stimulated spleen cells. As
shown in Fig. 7B (right panel) higher levels of
IFN- expression correlated with the higher T cell proliferation. The
findings shown in Fig. 7 established that the dose of 2 x
104 PFU of rVVlucIL-12 significantly increased the cellular
immune response against the gp160 Ag delivered by 1 x
107 PFU of rVVenv HA-, augmenting the numbers
of specific CD8+ T cells and specific Th1 cell
response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in serum.
To our knowledge this is the first study of IL-12 induction during VV
infection in mice. We have shown that IL-12 is produced after VV
infection as early as 6 hpi, and this is followed by an increase in
IFN- at 12 hpi (Fig. 3). This finding, observed at the protein level
in serum samples and at the mRNA accumulation in spleen cells, clearly
shows that VV per se induces a Th1-type immune response. Furthermore,
when IL-12 is expressed from the rVV, both the levels and time course
of IL-12 and IFN- accumulation are greatly enhanced following viral
infection.
The rVV expressing the IL-12 was attenuated, and it was eliminated from
target tissues at earlier times than control virus. By the second and
third dpi the levels of replication of rVV-IL-12 in spleen and ovaries
were 100-fold lower than those in mice inoculated with the
corresponding control virus. However, minor differences at the
anatomical level were observed in mice inoculated with the rVV
expressing IL-12. Splenomegaly was apparent in 70–80% of the mice
inoculated with 5 x 107 PFU of rVVlucIL-12, and this
was associated with a marked increase in levels of IFN- in serum
(data not shown), with no consequences on mouse survival during the
course of the experiment. These results agree with previous findings
after in vivo administration of rIL-12 (39).
Data obtained with in vitro restimulation of splenocytes from immunized
mice revealed that a Th1-type immune response is elicited upon
infection with VV. Moreover, after delivery of IL-12 from rVV, a
down-regulation of a Th2-type response is triggered (Fig. 4C). In this regard, we have found that VV infection elicits
an Ab response biased toward the isotype IgG2a, and the IgG2a/IgG1
ratio is increased 13.5 times relative to that for control VV when the
IL-12 gene is expressed from rVV. Hence, our findings clearly
demonstrate that IL-12 expression from rVV steers a potent Th1 response
following inoculation in mice.
Th1-type immune responses characterized by production of IFN- are
documented to occur during viral infections, pointing out the important
antiviral role played by this cytokine as a first-line defense
mechanism of the organism. A number of studies have observed IL-12
induction in mice upon viral infection. Expression of IL-12 has been
demonstrated in mice early after infection (12–24 hpi) with RNA
(murine hepatitis virus, lactate dehydrogenase-elevating virus,
influenza virus) and DNA (adenovirus, HSV-1, and murine CMV) viruses,
at both mRNA and protein levels (40, 41, 42). Studies on experimental
infection in mice with different viruses correlated the IL-12 effect on
antiviral immunity with the IFN- induction (43, 44) and activation
of the CTL response (45). However, alternative pathways of IFN-
induction during viral infection independent of IL-12 have been
reported (46). In mice inoculated with VV, it has been shown that the
anti-VV activity of IL-12 is abolished in IFN-R-/-
mice (19). Moreover, a drastic impairment of VV control results from
neutralization of IFN- or from the functional deficiency of the
IFN- gene in knockout mice (47). Thus, it is well established that
IFN- is involved in the clearance of VV infection. In view of our
findings, we propose that the rapid elimination of rVV expressing IL-12
in mice, compared with control virus infection, is mediated by the
induction of IFN-, acting as a first antiviral nonspecific immune
response of the host. Inhibition of VV replication by IFN- is
probably mediated by the production of nitric oxide, as we have
previously reported that treatment of macrophages with this cytokine
potently induce nitric oxide, and this correlates with inhibition of VV
replication (48). Furthermore, we have shown that inducible expression
of nitric oxide synthase by rVV leads to inhibition of VV DNA
replication and induction of apoptosis (49, 50).
As documented here, IL-12 expression by VV has the capacity to modulate
the antiviral immune response of the host, which was revealed by the
nearly 2-fold increase in the number of anti-VV CD8+
IFN--secreting cells at 7 and 14 days after inoculation compared
with that in controls. Although studies in other viral model systems
showed that IL-12 can promote unspecific expansion of CD8+
lymphocytes that can control viral infection (51), here we demonstrate
that a reduction in VV titers after expression of IL-12 correlated with
an increased number of specific CD8+ T cells. In
concordance with our results, expression from VV of IL-4, a cytokine
that down-regulates Th1 responses, inhibits the development of mature
antiviral CTL (52). Furthermore, mucosal delivery of IL-12 from rVV was
effective in restoring the antiviral CTL activity in a murine model of
allergic airway disease (28).
Different lines of evidence support the current opinion that induction
of CMI may be an important requirement for any candidate vaccine for
HIV-1. In this investigation we have examined the potential enhancement
of cellular immunity against HIV-1 by immunomodulating the specific
immune response to an HIV-1 Ag through the codelivery of IL-12 and the
HIV-1 Env protein. We found that immunization of mice with rVV
coexpressing both genes enhanced the cellular immune response against
Env when the rVV was administered at a dose of 1 x
107 PFU/animal, while a higher dose reversed the effect. We
found that the level of IFN- produced compromises the effectiveness
of the CMI elicited, as that parameter is critical in the velocity of
the resolution of the VV infection. However, expression of IL-12 to
levels above those induced as a consequence of the VV infection is
required to trigger a strong cellular anti-Env immune response.
These findings suggested that to achieve an enhancement of the cellular
immune response to HIV-1 Env, critical Ag and IL-12 expression levels
should exist. Indeed, in the SIV macaque model a direct correlation has
been shown between the ability of attenuated SIV to replicate in the
host and the degree of protection that was conferred (53). In this
regard, Orange et al. (51) showed that IL-12 doses required to promote
protective, but not detrimental, responses might vary extremely in the
context of different infections or immune responses.
We have also optimized the immunization procedure using the rVV
expressing IL-12. Thus, we found that delivering Env Ag and IL-12 genes
from individual VV vectors can lead to an enhancement of the specific
CMI to Env by using different doses of the two rVV. For this concern it
is noticeable that the dose-response effect of the IL-12 on the CMI
elicited is observed either using double rVV (Fig. 6) or delivering the
Ag and the IL-12 from separate vectors (see Fig. 7). However the
optimal dosages required are different in each case; thus, an
IL-12-mediated enhancement of the CMI anti-gp120 is observed at
107 PFU of the double rVV (expressing IL-12 and
env genes; Fig. 6, left panel), while in the
mixing experiment this effect was observed with 2 x
104 PFU of the IL-12-expressing rVV but not when 2 x
106 PFU was used (Fig. 7A). These empirical data cannot be
attributable to differences in IL-12 expression from the different rVV
(see Fig. 1B), but probably reflect the fact that
nonidentical infectious process are taking place in each case,
involving mechanisms that have a critical role in the balance between
the amount of IL-12 and the Ag. The finding that the dosage of rVVIL-12
plays a critical role in the generation of an effective Th1-type immune
response against the recombinant Ag is important, especially when
considering the implementation of vaccination strategies based on rVV.
In a recent vaccine trial with NYVAC-SIV recombinants in macaques, it
was found that although the addition of rNYVAC-IL-12 enhanced the CMI,
it did not appear to influence the outcome of SIV challenge (54). It is
possible that in this particular experimental animal model, the levels
of IL-12 expressed by rVV were not optimal for providing the
qualitative and/or quantitative modulation of the immune response to
influence the vaccine efficacy, as only one dose of the cytokine
delivering recombinant virus was assayed. Attempts to enhance the CMI
response against HIV-1 Ags by coadministration of IL-12 and plasmids
encoding HIV-1 genes have been described in various immunization
strategies based on DNA vaccines (55, 56, 57), and increases in both
specific CTL and Th1 proliferative activities were obtained (58). In
our study we demonstrate similar IL-12 effects when the cytokine is
delivered from VV vector, with the advantage that IL-12 is produced
only transiently, thus diminishing the undesirable side effects derived
from the expression of the cytokine for long periods of time, as
expected after DNA immunization.
In conclusion, in this investigation we have designed protocols of immunization based on rVV that increased significantly the cellular immune response to HIV-1 Env. We have also characterized the replication of rVV in tissues and how the IL-12 cytokine modulates the immune response to VV Ags. The feasibility of practical strategies able to enhance the immune response to an Ag delivered by rVV together with IL-12 and to steer the response toward a desired arm of the immune system, cellular or humoral, should provide a practical means to improve vaccination against pathogens, such as HIV-1, in which some immune responses may be protective and others detrimental.
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Acknowledgments |
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Footnotes |
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2 M.M.G. and J.C.R. contributed equally in the realization of this work.
3 Address correspondence and reprint requests to Dr. Mariano Esteban, Centro Nacional Biotecnologia, Campus Cantoblanco, 28049 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: VV, vaccinia virus; CMI, cell-mediated immunity; HA, hemagglutinin; hpi, hours postinoculation; IRES, internal ribosomal entry site sequence; dpi, days postinfection; HPRT, hypoxanthine phosphoribosyltransferase; PBS-T, PBS plus 0.05% Tween-20; ELISPOT, enzyme-linked immunospot.
Received for publication December 22, 1998. Accepted for publication March 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor. Science 259:1742.. Lymphokine Cytokine Res. 10:353.[Medline]
and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944. interferon are attenuated for nude mice. Proc. Natl. Acad. Sci. USA 89:3409. in interleukin 12-induced pathology in mice. Am. J. Pathol. 147:1693.[Abstract]
production and antiviral defense. J. Immunol. 156:1138.[Abstract]
interferon. J. Virol. 69:8147.[Abstract]
severely inhibits DNA synthesis of vaccinia virus in a macrophage cell line. Virology 198:731.[Medline]
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). The DNA cassette containing the genes
coding for IL-12 was inserted into the HA locus (
) to generate the
double recombinants, rVVlucIL-12 and rVVenvIL-12. p11, p7.5, and e/l
represent different VV promoters. B, Western blot
analysis of extracts from cells infected with the different rVV.
Monolayers of BSC-40 cells were infected (10 PFU/cell) with the
indicated rVV. The proteins were fractionated by SDS-PAGE under
reducing conditions, transferred to nitrocellulose paper, and reacted
with an anti-gp120 rabbit polyclonal Ab (left panel)
or an anti-IL-12 p40 rat mAb (right panel). Ab
reactivity was detected by immunoperoxidase staining using standard
procedures.
), or rVVlucIL-12 (•), and ovaries and spleens were collected on
the indicated days. The extent of virus gene expression was evaluated
by the luciferase assay as described in Materials and
Methods. Luciferase activity in the different homogenate tissue
samples was measured, and values were related to the amount of protein
present in tissue extracts (relative luciferase units (RLU) per
milligrams of protein). Background levels found in control uninfected
tissues were 3 and 4 log10 (RLU per milligrams of protein)
for spleen and ovaries, respectively. Results represent mean values
from samples of three animals per day and group with the SD. Similar
results were obtained in two independent experiments.
