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*
Department of Experimental and Diagnostic Medicine, Section of Microbiology, and Interdepartmental Center for Biotechnology, University of Ferrara, Ferrara, Italy; and
Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HIV-1-regulatory proteins Tat and Rev, as well as the accessory proteins Nef, Vif, Vpr, and Vpu, are considered attractive targets for the development of a multicomponent vaccine against HIV-1 infection. These proteins are well conserved among different isolates and thus may be less susceptible to mutations, as compared with structural genes, leading to production of escape virus variants (18, 19). In addition, they play a critical role in virus gene expression and replication and in the pathogenesis of AIDS and are immunogenic (1, 20, 21, 22). Among the regulatory genes, tat appears to be one of the most interesting targets.
The Tat protein is synthesized early during infection, and it is essential for virus replication (23). Tat is released by HIV-1-infected cells in the absence of cell death both in vitro and in vivo (24, 25, 26, 27, 28). Extracellular Tat can be taken up by HIV-1-infected cells, migrates to the nucleus, and trans activates the expression of the HIV-1 genome through autocrine and paracrine loops (25, 28, 29, 30, 31, 32, 33, 34, 35). In addition, extracellular Tat induces expression of the HIV-1 coreceptors on the target cells promoting virus spreading (36, 37) and exerts several effects on different types of uninfected cells (38), thus contributing to the pathogenesis of several AIDS-associated diseases including a severe form of Kaposi’s sarcoma, the most frequent tumor arising in AIDS patients, and lymphoproliferative and neurological disorders (24, 25, 26, 27, 39, 40, 41, 42, 43, 44, 45).
Tat is a 86–102-aa protein encoded by two exons. The first exon is conserved among different viral isolates and contains four functional domains including the amino-terminal (aa 1–21), the cysteine-rich (aa 22–37), the core (aa 38–48), and the basic (aa 49–72) domains. The cysteine-rich region represents the Tat trans activation domain. The basic region contains the nuclear localization signals and the binding sequences to the Tat-responsive element (23). In addition, the basic region and the RGD sequence of the second exon are required for the interaction of extracellular Tat with cell surface molecules (28, 32) and for Tat uptake by the target cells.
Abs against Tat functional domains as well as a CTL response specific to Tat have been detected in humans (8, 46, 47, 48). Ab titers and early CTL anti-Tat were shown to correlate with nonprogression to AIDS (8, 48, 49). In addition, in vitro studies have demonstrated that anti-Tat Abs neutralize the activity of extracellular Tat on HIV-1 replication, cell functions, angiogenesis, tumor promotion, and formation of metastases (24, 25, 26, 48, 50, 51). Altogether these observations suggest that the induction of humoral and cellular immune responses against Tat would likely induce, in addition to a prophylactic effect, an immunotherapeutic activity in HIV-1-infected patients, possibly extending the asymptomatic period of the disease.
The ability of the wild-type tat gene, as well as of the Tat protein, to evoke a broad immune response has been recently shown in mice (52, 53) and in humans (54, 55). However, the long term effects of the constitutive expression of a viral gene following genetic immunization are presently unknown. Therefore, in view of the possible use of the tat gene as a component of an anti-HIV-1 vaccine (prophylactic and/or therapeutic) and considering the possibility that the long term expression and release of wild-type Tat in vivo may determine reactivation of HIV-1 infection and other pathogenic effects in seropositive individuals, we have investigated two tat genes mutated in the trans activation domain (tat22 and tat22/37) as to their capability of eliciting an immune response against the wild-type Tat protein in the mouse model. The tat22 and tat22/37 genes were chosen because they lack HIV-1 trans activation activity and display a transdominant negative phenotype (56, 57). Therefore, the mutated protein products can also block the trans-activating activity of wild-type Tat protein which is produced in infected individuals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pC vector containing the hCMV IE3 promoter was derived from the plasmid pGEM7Zf(-) (Promega, Madison, WI). The 825-bp fragment, containing the hCMV IE promoter, was obtained by BamHI-ClaI digestion of the LNCX vector (58) and cloned into the BamHI-ClaI sites of pGEM7Zf(-). Plasmids pC-tat, pC-tat22, and pC-tat22/37 were constructed following insertion of the HIV-1 tat, tat22 (Cys22 to Gly), and tat22/37 (Cys22 to Gly, Cys37 to Ser) cDNAs into the pC vector. Briefly, the SalI-SmaI fragments of 360 bp containing the full-length cDNAs of HIV-1 (HXBC2 clone) tat, tat22, or tat22/37 were derived from the plasmids pTZ18U-tat, -tat22 or -tat22/37 previously described (56), filled in with Klenow enzyme, and inserted into the SmaI site of the polylinker region downstream to the hCMV promoter in the pC vector. The orientation of the cDNAs was determined following digestion with BamHI which cuts at nucleotide 356 in the SalI-SmaI fragments containing the tat genes. The reporter plasmid pU3RCAT, where expression of the CAT gene is driven by the HIV-1 LTR promoter, has been previously described (56). Plasmid DNAs were purified on CsCl gradients, according to standard procedures (59), and resuspended in sterile PBS, without calcium and magnesium.
Cell cultures and transfections
Jurkat T cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% heat-inactivated FBS (Life Technologies). Monolayer cultures of murine BALB/c 3T3 fibroblasts were grown in DMEM (Life Technologies) supplemented with 10% heat-inactivated FBS. The Superfect reagent provided by Qiagen (Hilden, Germany) was used for transient transfection of 5 x 105 BALB/c 3T3 fibroblasts, according to the manufacturer’s instructions. Cells were transfected with the pU3RCAT plasmid (1 µg) alone or in combination with pC-tat (1 µg). To assay for functionality of the tat mutant vectors, cells were cotransfected with pU3RCAT (1 µg), pC-tat (1 µg), and pC-tat22 (10 µg) or pC-tat22/37 (10 µg) vectors. The DEAE-dextran technique was used for transient transfections of 107 Jurkat cells (59). CAT activity was measured 48 h after transfection with amounts of cell extracts normalized to total protein content (59).
DNA immunization
Six-week-old female BALB/c mice (Nossan, Milan, Italy) were immunized with the plasmids, containing the tat22 or the tat22/37 gene. Animals were then tested for the development of humoral and cellular responses to wild-type Tat protein. Control animals included mice injected with the pC-tat or the empty vector pC. Each experimental group contained 10 animals. The dose of 100 µg of plasmid DNA in 100 µl was given to each mouse by bilateral i.m. injections in the quadriceps muscles of the posterior legs (day 0). The same dose of DNA was administered at days 15 and 30. Two blood samplings were taken at days 45 and 75 (bleedings 1 and 2), respectively. Mice were boosted three more times (at days 90, 120, and 150) with 50 µg/50 µl/animal of the plasmids injected i.m. in the quadriceps muscle of the right posterior leg. One week before the last two DNA immunizations, 100 µl of 0.5% bupivacaine hydrochloride (Sigma, St. Louis, MI) in isotonic NaCl were administered i.m. into the site of DNA inoculation. Blood drawings were taken 2 wk after each DNA injection at days 105, 135, and 165 (bleedings 3–5), respectively. Blood samples were withdrawn by endocardiac puncture from mice anesthetized with a mixture containing 30 µl of Ketavet-100 (Gellini Farmaceutici, Aprilia, Italy), 30 µl of Rompun (Bayer, Milan, Italy), and 30 µl of isotonic solution. Sera were prepared by centrifugation of coagulated blood at 8000 rpm for 5 min and stored at -70°C.
Tat protein expression and purification
The Tat protein was expressed in Escherichia coli and isolated by successive rounds of high pressure chromatography and ion-exchange chromatography as previously described (25, 28). The purified Tat protein is >95% pure as tested by SDS-PAGE and has full biological activity as tested by the rescue assay described below. The Tat protein was stored lyophilized at -70°C and resuspended in degassed PBS containing 0.1% BSA and 0.1 mM DTT before use. The plasticware was previously rinsed in PBS-BSA buffer for each procedure involving the use of Tat.
Tat protein immunization
Two 8-wk-old female BALB/c mice were immunized i.p. with wild-type Tat protein (10 µg/mouse) that was mixed with an equal volume of CFA (Sigma). Ten days later, each animal was boosted with 5 µg of protein mixed with an equal volume of in CFA. Two control mice were given the adjuvant alone. Ten days after the second immunization, the animals were sacrificed to collect blood and spleens.
Anti-Tat serology
The presence of anti-Tat-specific IgG in the sera of the
vaccinated animals was determined by ELISA. The Ag was a recombinant
maltose-binding protein (MBP) fused to Tat (MBP-Tat). The MBP-Tat
fusion protein was expressed in E. coli by means
of the pMal-c2 expression vector (New England Biolabs, Beverly, MA),
containing the full-length wild-type HIV-1 (HXBC2) tat cDNA, and
purified according to manufacturer’s instructions. The purity and
integrity of MBP-Tat was controlled by SDS-PAGE followed by Coomassie
blue staining. In the fusion protein, Tat corresponds to 
25% of the
total protein. Control Ag represented by MBP alone was also used. The
concentration of Ags were 5 µg/ml for MBP-Tat and 3 µg/ml for MBP
diluted in 0.1 M carbonate buffer (pH 9.6). Ninety-six-well
immunoplates (Nunc, Naperville, IL) were coated with 100 µl/well of
each Ag, sealed, and incubated for 16 h at 4°C. Plates were
washed three times with PBS (pH 7.4) containing 0.05% Tween 20
(Sigma). Sera were diluted in carbonate buffer and tested in duplicate.
Fifty-microliter aliquots were added to each well and incubated for 90
min at 37°C. Plates were washed three times with PBS/Tween 20 buffer
before addition of 100 µl of horseradish peroxidase-labeled goat
anti-mouse IgG (Bio-Rad, Richmond, CA) diluted 1/1000 in PBS
without calcium and magnesium and containing 0.1% Tween 20 and 1%
BSA, and incubated for 90 min at room temperature. Following three
washes with PBS/Tween 20, 100 µl of ABTS substrate (Boehringer
Mannheim, Mannheim, Germany) were added for 45 min at room temperature.
The absorbance was measured at 405 nm. The cutoff value for DNA and
protein immunization corresponds to the mean absorbance value (+2 SD)
of all the control sera derived from animals immunized with the empty
pC vector or the adjuvant alone. Absorbance values higher than the
cutoff were considered positive.
Epitope mapping of anti-Tat Abs and Ab isotyping
For epitope mapping, eight synthetic peptides (synthesized at the Department of Pharmaceutical Science, University of Ferrara, Ferrara, Italy), representing different regions of Tat, were used at the concentration of 10 µg/ml. After coating with 100 µl/well, plates were blocked with 100 µl/well of PBS with 3% BSA for 2 h at 37°C, washed three times, and incubated with 50 µl/well of each serum, diluted in PBS with 3% BSA. The assays were then performed as described above.
For isotyping, plates were coated with MBP-Tat and incubated with the mice sera as described above. After washing, 100 µl of goat anti-mouse IgG1, IgG2, IgG2a, IgG2b, or IgG3 (Sigma) were added to the wells and incubated for 90 min at room temperature. Immunocomplexes were detected with a horseradish peroxidase-labeled rabbit anti-goat (Sigma) and ABTS substrate as described above.
Neutralization assays
For neutralization assays, HLM1 cells, a HeLa-CD4+ cell line containing an integrated copy of a tat-defective provirus, were seeded in 24-well plates at a density of 6 x 105 cells/well in DMEM supplemented with 10% inactivated FBS. After 24 h, cells were washed with PBS, and Tat protein (30 ng/ml) was added alone or after prior incubation with an equal volume of the mouse serum, as previously described (25, 28). After 48 h, the rescue of HIV-1 replication was monitored in the culture supernatants by using a p24 Ag capture assay (NEN-DuPont, Stevenage, U.K.).
Tat-specific T cell proliferation
Mononuclear cells were purified from spleens of sacrificed animals by Ficoll-Histopaque 1083 (Sigma) density gradients, washed twice with PBS, and resuspended at 2 x 106/ml in RPMI 1640 supplemented with 10% heat-inactivated FBS. Cells were counted by trypan blue exclusion dye and cultured at 2 x 105/well in 200 µl in the presence of affinity-purified Tat protein (0.1, 1, or 5 µg/ml) or Con A (10 µg/ml, Sigma) for 5 days. [methyl-3H]Thymidine (2.0 Ci/mmol, NEN-DuPont) was added to each well (0.5 µCi), and cells were incubated for 16 h. [3H]Thymidine incorporation was measured with a ß-counter. The SI was calculated by dividing the mean counts/min of six wells of Ag-stimulated cells by the mean counts/min of six wells of the same cells grown in the absence of the Ag. A value >2.3-fold SI that corresponds to the mean SI of the control mice immunized with the pC vector (+2 SD) was considered positive.
Statistical analysis
Student’s t and 
2 tests were
performed as described (60).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, pC-tat
expressed a functional Tat protein after transfection into murine
BALB/c 3T3 fibroblasts, as detected by the activation of the reporter
plasmid pU3RCAT, where expression of the CAT gene is driven by the
HIV-1 LTR (Fig. 1A, lane 1). Similarly, the
plasmids pC-tat22 and
pC-tat22/37 expressed transdominant negative
mutants capable of competing wild-type Tat activity on the HIV-1
LTR-CAT vector after transfection of pC-tat with a 10-fold
molar excess of each mutant plasmid (Fig. 1A, lanes
2 and 3). Similar results were obtained in human
Jurkat T cell lines (data not shown).
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B). Control mice were immunized with pC-tat or
the empty pC vectors. All animals in each group were injected three
times (days 0, 15, and 30) with 100 µg of each plasmid DNA. Two weeks
after the third immunization (day 45), a humoral response to wild-type
Tat was detected in 20 and 30% of mice injected with
pC-tat22 or pC-tat22/37,
respectively, as well as in 60% of control animals vaccinated with
pC-tat. However, a slight decrease in the percentage of
positive animals was observed in almost all groups 4 wk later (day 75)
(Table I). There was no significant
difference (p > 0.05) between the percentage
of positive animals in the groups immunized with tat mutants
and wild-type tat. All mice were then boosted three more
times with 50 µg of the plasmid DNAs (days 90, 120, and 150). In
addition, since bupivacaine has been shown to increase the uptake of
DNA from muscle cells and enhance the response to DNA immunization
(16), 1 wk before the last two immunizations the animals were given
bupivacaine at the site of inoculation. Following these additional
boosts and the bupivacaine treatment, the percentage of responder
animals increased to 100% in the group immunized with
pC-tat22 as well as in the control mice
receiving pC-tat, and to 85% in the group boosted with
pC-tat22/37 (bleeding 5 at day 165) (Table I).
Ab titers increased from a mean value of 1/50, detected at the first
bleeding, up to 1/200–1/3200 at the fifth bleeding (Table II). All anti-Tat-positive sera did
not bind the bacterial MBP Ag alone, confirming that the reactivity
to MBP-Tat used as Ag was specific to Tat (Table II). To compare
the results of DNA immunization with protein immunization, two mice
were immunized with the Tat protein. They were injected twice (days 0
and 10) with the Ag and sacrificed 10 days after the second
immunization. In both animals, anti-Tat-specific Abs were detected
with titers of 1/6400 (Table II).
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A). A second reactive epitope
was identified at residues 73–86 in sera of
pC-tat22 and pC-tat immunized
animals, and at amino acids 52–72 in
pC-tat22/37-immunized mice (Fig. 2A).
Epitopic sites mapping at amino acids 1–20 and 21–40 were instead
recognized by mice injected with the Tat protein (Fig. 2B).
The isotype analysis of the IgG subclasses indicated a prevalence of
IgG2a in mice vaccinated with both the mutated and the wild-type
tat genes (Fig. 3), whereas
IgG1 and IgG3 isotypes were detected in animals immunized with the Tat
protein (data not shown).
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A proliferative response to Tat was evaluated on animals splenocytes in
the presence of Tat. A specific response was exhibited by animals
inoculated with pC-tat22 or
pC-tat22/37 plasmids, as well as by the control
mice treated with pC-tat DNA or the Tat protein (Table III). Splenocytes from several mice
responded to as little as 0.1 µg/ml recombinant Tat (data not shown);
however, the highest responses were seen after stimulation with 1 and 5
µg/ml of Tat. Although the highest frequency (87%) of animals
showing a proliferative responses to Tat was observed in the group of
mice immunized with wild-type tat DNA, a similar
(p > 0.05) T cell response specific to Tat was
elicited, in the same range of SI, in 50 and 71% of mice immunized
with the tat22 and
tat22/37 plasmids, respectively (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, we have considered that constitutive expression of the tat gene following genetic immunization may induce HIV-1 replication and eventually exerts pathogenic effects when administered to humans. Thus, we have elicited and analyzed the immune response toward the wild-type Tat protein that is evoked by tat22 and tat22/37 genes. These genes are mutated in the trans activation domain of Tat and therefore lack Tat activity. In addition, they block the effect of wild-type Tat on the HIV-1 LTR.
Our results demonstrate that these tat mutant genes induce humoral and cellular immune responses specific to the wild-type Ag. The humoral response was readily detected after the third immunization with each gene. The mean titers and the number of responder mice increased following three additional boosts and treatment with bupivacaine that was previously shown to facilitate the uptake of plasmid DNA by the muscle tissue (16). The Ab titers, the IgG isotypes, the Tat epitopes recognized by the Abs, the capability of animal sera to neutralize the rescue of HIV-1 replication by exogenous Tat, and the T-cell proliferative responses were comparable (p > 0.05) in the three groups of mice immunized with the mutant or the wild-type tat genes. In addition, after vaccination with the tat genes, Abs reacted with a broad spectrum of linear Tat epitopes, mapping at residues 21–40, 52–72, and 73–86, and the prevalence of IgG2a subclass was suggestive of a Th1 profile.
A broad immune response, both humoral and cellular, was also detected after protein immunization. In these animals, however, anti-Tat Ab titers were higher, and consequently the rescue of Tat-induced HIV-1 replication was blocked more effectively than by sera from DNA-immunized mice. Finally, in these mice the epitope reactivities were more restricted, and the presence of IgG1 and IgG3 isotypes was suggestive of a Th2-like response. These differences between genetic vs protein immunization with tat may be explained by a diverse conformation of the native Tat protein expressed in vivo, as compared with the exogenous recombinant protein injected into the animal. Indeed, because of its high content in cysteine residues, the Tat protein is very labile when exposed to air and light, and it is sensitive to temperature (28). In addition, Ags produced by plasmid DNA administered i.m. might be presented less efficiently to the immune system by MHC class I molecules on myocytes, whereas a soluble Ag injected by the i.p. route may be readily presented to T cells in the spleen or in the local lymph nodes. Another reason for the differences observed is that most of the Tat molecules, released by the injected myocytes, may be locally retained and hardly reach the immune compartment.
The results of this study demonstrate that both arms of the immune system can be primed by tat22 and tat22/37 genes that induce a broad response specific to the wild-type Tat protein and similar to that elicited by the wild-type tat DNA. These results show that genetic vaccination with tat22 and tat22/37 induces a Th1-like response similarly to that found with wild-type tat, by us in this study and by others previously (53). The data presented also indicate that neutralizing anti-Tat Abs raised by tat-mutated genes can block the effect of the extracellular Tat protein on virus replication. These results are encouraging for the development of an anti-HIV-1 vaccine based on Tat. In fact, Tat-neutralizing Abs may control virus replication and the spreading of infection (8, 48, 49, 50). In addition, a Th1 response may be the most effective in controlling the progression of HIV-1 infection to AIDS (64, 65, 66, 67).
Several questions, however, remain to be settled and represent the matter for further studies. For instance, the cysteine substitutions in tat22 and tat22/37 gene mutants fall just within the aa 21–40 epitope which is immunodominant in DNA immunization and in HIV-1 natural infection (22, 46, 47). It would be interesting, therefore, to compare the immune response to the Tat protein and to Tat 21–40 peptide mutated in the same cysteine residues, although the results may not be directly comparable because the immunodominant epitope in protein immunization is at aa 1–20. Also, it is not clear at present whether a genetic or a protein immunization to Tat would be more protective in natural HIV-1 infection. From the results of this study, a DNA immunization seems to be preferable, due to the presence of a cellular response with the characteristics of a Th1 reaction. Moreover, it would be now of great interest to perform an alternate prime-and-boost experiment, where mice will be primed with either tat DNA or Tat protein and then boosted with the other. In this study, we have intentionally avoided boosting with the Tat protein the animals immunized with the tat genes, because our aim was to characterize the response of mice to a pure tat DNA immunization. Comparing the results of these experiments to the results obtained by immunization with only tat DNA or Tat protein would provide useful information and suggestions to establish an effective program for Tat immunization (genetic and/or protein) in natural HIV-1 infection. In addition, the extension of these studies to the animal model of nonhuman primates which are susceptible to virus infection will determine the effect of vaccination with Tat on protection from infection and from disease progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Antonella Caputo, Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, Via Luigi Borsari 46, I-44100 Ferrara, Italy. E-mail address:
3 Abbreviations used in this paper: hCMV IE, human cytomegalovirus immediate early promoter; CAT, chloramphenicol acetyltransferase; SI, stimulation index; LTR, long terminal repeat; MBP, maltose-binding protein.
Received for publication November 12, 1998. Accepted for publication February 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and -
and -
isoforms in PC12 cells. Biochem. Biophys. Res. Commun. 242:332.[Medline]
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