A Monoclonal Antibody Directed against a Conformational Epitope of the HIV-1 Trans-activator (Tat) Protein Neutralizes Cross-clade

Background: Tat, an essential HIV-1 replication factor and extracellular toxin acting on immune cells, is an interesting target to develop therapeutic antibodies. Results: mAb 7G12 recognizes a conformational epitope of Tat, cross-neutralizes different Tat variants, and blocks Tat uptake. Conclusion: mAb 7G12 could be used in immunotherapy to restore the immunity of patients. Significance: Development of alternatives to antiretroviral therapy is crucial. The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.

The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.
Developing an effective vaccine against HIV-1 remains an important task because of the high genetic variability of HIV-1. In addition, the virus capability to evade immune responses, particularly in immunologically weakened infected patients, highlights the challenge to find a target that induces broadly reactive antibodies. Besides, an efficient vaccine must elicit potent HIV-1 neutralizing antibodies. Conserved epitopes are generally considered to be the key approach to obtain such broadly neutralizing antibodies. Passive immunization studies with mAb targeting the Env protein in simian-human immunodeficiency virus challenge showed promising results (1). However, other potential HIV-1 protein targets such as transactivator of transcription (Tat) 3 should be considered because of its extracellular functions. Tat is one of the first proteins produced by infected cells (2). It is essential for initiation (3) and elongation (4) of HIV-1 gene expression. Moreover, Tat is secreted from HIV-1 infected cells and can cross membranes, inducing apoptosis in different immune cells and protecting HIV-1-infected cells and reservoir cells against the cellular immune system (5). Extracellular Tat also plays a role in spreading the infection by inducing the expression of chemokine receptors CCR5 and CXCR4, which are CD4 coreceptors for HIV-1 (6).
A clade B Tat variant, named Tat Oyi, has been cloned in the 1980s from a seropositive patient who did not develop AIDS in a remote area of Gabon. HIV-1 Oyi genes were similar to genes of usual HIV-1 strains except the tat gene, which had mutations never found in other Tat variants (7). We showed previously that rabbit immunization with the Tat Oyi variant rose antibodies able to recognize different Tat variants (8). A heterologous simian-human immunodeficiency virus-BX08 challenge carried out on macaques vaccinated with Tat Oyi showed a reduced viremia in vaccinated monkeys. Furthermore, reservoir cells were no longer detectable (9). Thus, Tat Oyi has specific immunogenic features to generate neutralizing mAbs against Tat variants (8).
In this study, we immunized mice with Tat Oyi and screened mAbs for their cross-clade recognition. We selected one IgG1 mAb, named 7G12, showing an efficient cross-recognition against various HIV-1 subtypes. mAb 7G12 was able to neutral-ize the biological activities of Tat variants from the five main HIV-1 subtypes and to block Tat uptake. This is the first report of a broadly neutralizing mAb against Tat with a therapeutic potential.

EXPERIMENTAL PROCEDURES
Tat Variants and Peptide Synthesis-Tat Oyi was assembled in solid phase synthesis as described previously (10). A Ser 3 Cys substitution at position 22 in Tat Oyi sequence (Fig. 1) allowed recovering biological activity of Tat Oyi and its use in neutralization assays with antibodies. Five peptides covering the full sequence with overlaps (1-22, 13-46, 38 -72, 57-86, and 72-101) and, respectively, named peptide 1 to 5 were synthesized. Other synthesized Tats correspond to clade A (Ug11RP), clade D (Eli), circulating recombinant form AE (CM240), clade C (96Bw), and clade B, predominant in Europe and the Americas (HxB2) (Fig. 1). Purification and analysis were performed as described previously (10). Purity and mass were controlled by mass spectrometry. After lyophilization, biological activity of Tat variants were checked by transactivation assays with HeLa P4 cells as described previously (11).
Immunization and Monoclonal Antibody Production-Four BALB/c mice were immunized with 10 g of synthetic Tat Oyi in 100 l of phosphate calcium gel adjuvant (Brenntag Biosector) by the subcutaneous route. Two weeks later, mice were boosted with the same preparation. Five weeks later, mice were boosted again with the same preparation by the intramuscular route. Three control mice were also immunized with the same adjuvant and protocol but without Tat protein. On day 45, mice were euthanized and bled terminally. The spleenocytes were immediately separated to hybridize with myeloma cells as described (12). Supernatants of isolated hybridoma were screened by ELISA against Tat Oyi variant to identify producing clones. Then, they were subcloned by limiting dilutions (Ͻ 1.0 cell/well) twice, and antibody positive clones were screened by ELISA against firstly Tat Oyi and then against Tats Ug11RP, ELI, CM240, 96BW and HxB2. Previously, a similar immunization with Tat Oyi of four rats has been performed and a mAb (IgG1), named 27A8, had been selected on its high recognition of Tat Oyi and was used as a control. Selected hybridomas were cultured, and mAbs were purified by protein G chromatography (Roche) according to the manufacturer's instructions. After purification, antibodies were dialyzed against Hepes buffer 20 mM, NaCl 120 mM (pH 7.3) and concentrated at 1 mg/ml.
Detection of Purified mAbs Responses by ELISA-96-well plates were coated with 100 ng of folded or denatured Tats or peptides 1 to 5 (1 g/ml in phosphate buffer 100 mM (pH 4.5)) or peptides pool (each peptide, 1 g/ml in phosphate buffer 100 mM (pH 4.5)). Tats (10 g/ml) were denatured by heating at 90°C during 20 min in presence of urea 3 M and then diluted 10-fold with cold phosphate buffer 100 mM (pH 4.5). To control urea effect, folded Tats were coated in the presence of 0.3 M urea. The nonspecific signal was controlled in coating 100 ng of BSA by well.
Following blocking with 5% skim milk and washing steps, plates were incubated with 100 l of diluted purified mAb for 1 h at 37°C. After the washing steps, 100 l of HRP-conjugated anti-mouse or anti-rat IgG (GE Healthcare) were added, and the plates were incubated for 1 h at 37°C. After washing steps, 100 l of 2.2Ј-azino-di (3-ethylbenzothiazoline-6-sulfonate) (ABTS) substrate (Roche) was added. The absorbance was measured at a wavelength of 405 nm 1 h later.
Measurement Association Rate Constants of mAb 7G12 with Tats in Solution-An ELISA-based method was used for measuring antigen/antibody association rate constants in solution (13). Briefly, 96-well plates were coated with 100 ng of Tat Oyi in 100 mM phosphate buffer (pH 4.5) overnight at 4°C. One well of four was left uncoated. Following washing steps, the plates were blocked with 5% skim milk in PBS for 1 h. The antibody (1 g/ml) and the Tat variants (0 to 0.4 g/ml) solutions were prepared in 20 mM Hepes buffer, 120 mM NaCl (pH 7.3) at twice the final concentration. At time zero, similar volumes of antibody and antigen solutions were mixed, and 100 l were immediately (time zero of the kinetic analysis) transferred to three coated wells and one uncoated well. Each 5 min, the wells were filled with a sample of mAb/Tat mixture. Each sample was incubated in the wells for 4.5 min and removed. At the end of the kinetic analysis, plates were washed and incubated with HRPconjugated anti-mouse IgG. After washing steps, 100 l of ABTS substrate was added. The absorbance was measured at a wavelength of 405 nm 1 h later. Association and dissociation rate constants (k on and k off ) were then determined, allowing the calculation of the equilibrium dissociation constant (K D ).
Western Blot Analysis-Tats (100 ng) were subjected to SDS-PAGE (15%) under reducing conditions (DTT 100 mM and urea 6 M in Laemmli sample buffer at 96°C for 10 min). Tats were then electrotransferred to nitrocellulose membrane (Schleicher and Schuell). After blocking with 5% skim milk, strips of the membrane were incubated for 1 h with mAbs 7G12 or 27A8 at 1:2500. The secondary antibody was HRP-conjugated anti-mouse or anti-rat IgG (GE Healthcare) diluted 1:1000, and bands were revealed with H 2 O 2 0.1%, diaminobenzidine tetra hydrochloride (Sigma) as substrate.
Neutralization of Tat Transactivation-Neutralization of Tat transactivation was performed with HIV long-terminal repeat (LTR) promoter transfected HeLa cells (HeLa-P4) and analyzed by monitoring the production of ␤-galactosidase (␤-Gal) as described previously (11). Briefly, HeLa-P4 cells were incubated in DMEM with glutamine supplemented with 10% (v/v) heat-inactivated fetal calf serum and 50 units/ml neomycin. 2 ϫ 10 5 cells/well were incubated in 400 l of DMEM supplemented with 0.01% (w/v) protamine (Sigma-Aldrich) and 0.1% (w/v) BSA (Sigma) in 24-well plates (Falcon). Lyophilized or denatured Tats were diluted at 5 M (or 10 M) in phosphate buffer 100 mM (pH 4.5). Tat (0.5 M final) and described concentrations of antibodies were added to cells. Volumes were completed to 500 l with 20 mM Hepes buffer, 120 mM NaCl (pH7.3). When added together, denatured, and folded, Tat had a final concentration of 0.5 M each.
After 18 h at 37°C, ␤-Gal production was measured with a ␤-Gal ELISA kit (Roche) according to manufacturer's recommendations. The background against BSA was removed. The transactivation ratio was the amount of ␤-Gal in the presence of Tat divided by the amount without Tat.
Methyl Thiazolyl Tetrazolium Assay-The Jurkat T-cell quantity remaining after incubation with Tat, reflection of induced apoptosis, was measured with an MTT-based method as previously described (14). Jurkat T-cells were cultivated in RPMI 1640 medium with glutamine supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 g/ml streptomycin, and 100 units/ml penicillin. 10 5 cells in 100 l of RPMI with 0.1% BSA and 0.01% protamine A were transferred in wells of 96-wells plates. Lyophilized Tats were diluted at 65 M in 100 mM phosphate buffer (pH 4.5). Cells were incubated for 48 h at 37°C with 10 l of Tats and 20 l of mAbs or 20 mM Hepes buffer, 120 mM NaCl (pH7.3). Control wells were adjusted with 10 l of 100 mM phosphate buffer (pH 4.5) and 20 l of 20 mM Hepes buffer, 120 mM NaCl (pH7.3) to verify cell viability. 0.5 mg of MTT (InterChim) was added and incubated for 5 h. Formazan crystals were dissolved in 200 l dimethyl sulfoxide, and absorbance was measured at 510 nm.
Tat Uptake-Translocated Tat localization in the nucleus and cytosol was evaluated by immunoblot analysis in Jurkat and HeLa cells. Jurkat T-cells (or HeLa cells) were incubated as described above. 10 6 cells by well (24-well plate) were incubated in 400 l of RPMI (or DMEM) supplemented with 0.01% (w/v) protamine (Sigma) and 0.1% (w/v) BSA (Sigma). Lyophilized Tats were diluted at 50 M in 100 mM phosphate buffer (pH 4.5). 50 l of Tat (5 M final) and 50 l of purified antibodies (50 g) or 50 l of 20 mM Hepes buffer, 120 mM NaCl (pH7.3) were added, and cells were incubated for 2 h at 37°C. Cells were washed with 1 ml PBS and potterized in lysis buffer (50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, and protease inhibitors (Roche)). Complete lysis was controlled by microscopy. A pellet corresponding to the nuclear extract was obtained by centrifuging the lysate at 600 ϫ g for 15 min at 4°C. Supernatant was centrifuged at 100,000 ϫ g for 1 h at 4°C, and the membrane pellet was retrieved. The cytoplasmic fraction (supernatant 2) was Trichloroacetic acid precipitated overnight at Ϫ20°C. The final pellet was washed by 1 ml of cold acetone. Nuclear, membrane, and cytoplasmic pellets were subjected to SDS-PAGE (15%) under reducing conditions (100 mM DTT and urea 6 M in Laemmli sample buffer at 96°C for 10 min) and electrotransferred to a nitrocellulose membrane (Schleicher and Schuell). Protein amounts were controlled by staining with Ponceau red (Sigma). After blocking with 5% skim milk, membrane was incubated overnight with an anti-Tat rabbit sera (1:1000) described previously (11). The secondary HRP-conjugated anti-rabbit antibody (GE Healthcare) was diluted to 1:5000, and bands were revealed with Immobilon Western chemiluminescent HRP substrate (Millipore). The intensity of the bands was analyzed by densitometric imaging using the freely available ImageJ program (National Institutes of Health). Densitometries in the nucleus and cytosol were added to evaluate total translocated Tat without antibody (100%). Densitometries of each compartment in the presence of antibodies were compared and expressed as a percentage. Annexin 1, P-AC-histone H3, and Fusin (Santa Cruz Biotechnology) antibodies were used as cytoplasmic, nuclear, and membrane fractions control, respectively.
Statistical Analysis-Statistical differences were analyzed by use of a Mann-Whitney test. p Ͻ 0.05 was considered significant.

mAb 7G12 Cross-recognizes Tat Variants from the Five Main
HIV-1 subtypes-Mice were immunized with Tat Oyi, and one IgG1 mAb, named 7G12, was selected among 132 prescreened clones for its broadly reactive immune response against a panel of Tat variants representative of main HIV-1 clades (Fig. 1). To characterize the cross-recognition, the affinities of mAb 7G12 for the different Tat variants were evaluated in ELISA ( Fig. 2A). mAb 7G12 bound all Tats with a similar high affinity. Only one mAb, named 6E7, also showed a broadly reactive immune response but with 10 times lower affinities (Fig. 2B). All other clones did not recognize all tats, showed very low affinities, or were IgM subtype. The control mAb named 27A8 bound only Tats Oyi and HxB2 with high affinities but not the others (Fig.  2C).
To quantify mAb 7G12 affinities for Tat variants, an ELISAbased method (13) was used to measure antigen/antibody association rate constants in solution. Antigen and antibody were mixed, and aliquots were withdrawn at different time intervals to determine the amount of free antibodies. The disappearance of free antibodies reflected the time course of the association reaction. Affinity constant obtained for Tat Oyi was high (K D ϭ 7 Ϯ 0.4 nM). K D for Tats UG11RP (12 Ϯ 0.5 nM), Eli (5.7 Ϯ 0.1 nM), CM240 (3.2 Ϯ 0.1 nM), and 96BW (7.4 Ϯ 0.3 nM) were very similar. These results confirmed ELISA affinity curves ( Fig. 2A) and suggested that mAb 7G12 recognized a common site on the surface of all Tat variants tested. Interestingly, sequence comparison shows only a small percentage of potential continuous epitopes (Ͼ 5 amino acids) between the different Tats (Fig. 1).
mAb 7G12 Recognizes a 3D Epitope-To map the epitopes recognized by mAbs 7G12, 6E7, and 27A8, ELISA was carried out as described previously (15), using 100 ng of different overlapping peptides spanning the entire sequence of Tat Oyi (Fig.  3A). mAb 7G12 did not recognize peptides, whereas mAb 27A8 recognized peptide 5 covering the C-terminal sequence (72 to 100). Thus, mAb 27A8 recognized a linear epitope in the C-terminal domain, conserved only between Tats Oyi and HxB2 (identity 87.1%) but not in other Tat variants. In contrast, mAb 6E7 recognized peptides 1 and 2, indicating that its epitope matched, at least partly, with the overlapping sequence (residue 13 to residue 22). Thus, mAb 6E7 showed a broad immune response against the panel of Tat variants because this linear epitope is highly conserved in the N-terminal domain of these proteins (Fig. 1). These data suggested that mAb 7G12 did not recognize a linear epitope but a 3D epitope on the surface of Tat Oyi that corresponds to a particular folding highly conserved in Tat variants.
To test this hypothesis, ELISA with mAbs 7G12 and 27A8 were performed against folded and denatured Tats Oyi and HxB2 (Fig. 3B). Urea alone, used for complete denaturation, did not modify the ELISA responses for both mAbs, whereas high temperature drastically decreased mAb 7G12 binding to Tat without modifying 27A8 binding. mAb 7G12 did not recognize Tat variants following denaturation or a peptide pool, highlighting that this antibody recognized a 3D epitope and not a linear epitope. In contrast, mAb 27A8 recognized denatured Tat variants and a peptide pool, confirming that this mAb was directed against a linear epitope. Moreover, mAb 7G12 was also unable to recognize Tat Oyi in Western blot analysis (Fig. 3C). In contrast, mAb 27A8 in the same dilution strongly recognized Tat Oyi.

mAb 7G12 Neutralizes Tat Oyi Transactivation Activity-
We studied the effects of mAb 7G12 (3D epitope) and of mAbs 6E7 or 27A8 (linear epitopes) on the Tat Oyi biological activities. The neutralizing effect of mAb 7G12 on transactivation activity of Tat Oyi was monitored with HeLa P4 cells (Fig. 4A). 50 g of mAb 7G12 neutralized almost completely Tat Oyi (0.5 M) transactivation activity. The neutralization potency of mAb 7G12 correlated with the concentration of antibody. In our assays, the maximal neutralization was reached with 0.33 nmoles (50 g) of mAb against 0.25 nmoles of Tat, suggesting a molar-molar association. In contrast, mAb 27A8 only had a very low effect on transactivation activity of Tat Oyi (Fig. 6A).
To determine whether the neutralizing effect of mAb 7G12 was due to recognition of a 3D epitope, transactivation activity was examined using 0.5 M of folded and denatured Tat Oyi (Fig. 4B). Denatured Tat Oyi had lost the transactivation activ-   APRIL 6, 2012 • VOLUME 287 • NUMBER 15 ity but did not prevent the biological effect of folded Tat when both were added together. Interestingly, unfolded Tat did not decrease the neutralizing effect of 50 g of mAb 7G12 when folded and denatured Tats were in competition. This demonstrated that the activity of Tat Oyi in solution was conformation-dependent and blocked by the mAb 7G12, which recognized a 3D epitope. mAb 7G12 Inhibits Tat Oyi-induced Apoptosis in Lymphocytes-Discordance between the transactivation and other Tat functions was observed previously (16). Thus, to confirm the mAb 7G12 neutralizing effect, an inhibition assay was performed on Tat-induced apoptosis (17). Viability of Jurkat lymphocytes cells was monitored in presence of Tat Oyi, using a tetrazolium salts-based colorimetric assay (Fig. 4C). 10 M of Tat Oyi almost completely inhibited formazan production in Jurkat cells. This inhibition was correlated with the concentration of Tat Oyi up to 1 M. The neutralizing effect of mAb 7G12 was evaluated (Fig. 4D). 20 g (1 M) of mAb 7G12 completely blocked the apoptosis of lymphocytes induced by Tat Oyi (5 M). Partial neutralization was observed up to 5 g (0.25 M) of mAb 7G12 but not with 1 M of mAb 27A8 (Fig. 6B).

Tat Broadly Reactive Neutralizing mAb
mAb 7G12 Blocks Extracellular Tat Uptake-Exogenous Tat is able to enter cells, inducing apoptosis or activating transcription. We evaluated the ability of mAb 7G12 to neutralize Tat Oyi uptake using Jurkat cells (Fig. 5). mAb 27A8, which recognized a linear epitope of Tat Oyi, was used as mAb control. As described previously (18,19), most internalized Tat was located in the nucleus (78%), and only a small part was in the cytosol (22%). mAb 27A8 only showed a non-significant decrease in cytosolic fraction. In contrast, mAb 7G12 almost totally blocked Tat translocation, as shown by the significant drop in the nuclear (-75.6%) and cytosolic (-98.5%) fractions. Very similar results were observed with HeLa cells (data not shown). Thus, mAb 7G12 was able to block Tat uptake, preventing apoptosis and transcription more effectively than a mAb that recognized only a linear epitope.
mAb 7G12 Cross-neutralizes Biological Activities of Other Heterologous Tat Variants-Cross-neutralizing activity of 50 g (0.66 M) of mAb 7G12 was evaluated using the transactivation assay with HeLa P4 cells and 0.5 M of Tat variants representative of the various HIV-1 subtypes including A, B, C, D, and the circulating recombinant form AE (Fig. 6A). MAbs 27A8 and 6E7 were used as control at the same concentration. mAb 7G12 inhibited the transactivation activity of all Tat variants as well as with Tat Oyi. Precisely, mAb 7G12 inhibited 61.9%, 66.3%, 57.1%, 53.2% and 50.8% of transactivation activities of Tats UG11RP, HxB2, 96Bw Eli, and CM240 respectively, compared with 63.7% observed with Tat Oyi. In contrast, mAb 27A8 showed a low inhibitory effect only for Tat Oyi and Tat HxB2 (12% and 19.4%, respectively) and no significant inhibition with others Tats. mAb 6E7 had no inhibitory effect on all Tat variants. We also examined the neutralizing effects of mAbs 7G12, 27A8, and 6E7 on inhibition of lymphocytes proliferation induced by 5 M of Tat variants (Fig. 6B). Unlike mAbs 27A8 and 6E7, mAb 7G12 completely reversed the proliferation inhibition induced by the Tat variants.
Thus, mAb 7G12 neutralized the biological activities of the different Tat variants tested with a similar efficiency, suggesting that the epitope recognized by this antibody was conserved on the surface of the variants and that their folding play an important role for Tat activity.

DISCUSSION
Up to now, the main targets to obtain therapeutic antibodies to HIV-1 were the surface envelope glycoprotein gp120 and the transmembrane glycoprotein gp41 (20 -28). These antibodies were designed to block virus uptake in cells, but resistances emerged by selection of escape mutants. Most of these antibodies recognize linear epitopes that can mutate easily or be modified after transcription (29 -31). Recently, screening strategies in infected patients using new technologies have selected neutralizing antibodies efficient against a large number of variants of HIV-1 (32)(33)(34). These studies highlight that only a very limited number of antibodies in a few patients are broadly neutralizing. Some mAbs against Tat were developed on mice (35)(36)(37) and tested on humans (38,39), but none of them had the capacity to cross-recognize Tat variants.
In this study, we used the immunogenic property of Tat Oyi to obtain a mAb, named 7G12, selected for its high recognition of several genetically heterologous Tat variants. This specific feature of Tat Oyi has been described previously (8). Among these variants, the Tat HxB2 sequence has the highest sequence identity (87.1%) with Tat Oyi, whereas Tat variants CM240, UG11RP, 96Bw, and Eli have identities of 67.3%, 65.3%, 67.3%, and 73.3%, respectively. Moreover, identical amino acids among the six variants are gathered in sequence between resi-dues 13 to 18 and residues 43 to 52 (Fig. 1). The sequence 13-18 corresponds to the core region of Tat, and the sequence 43 to 52 appears to lack immunogenicity, although exposed to solvent (40). mAb 6E7, used as a control, recognized a linear epitope located in the common sequence (residues 13 to 22) of the N-Terminus of Tat. mAb 6E7 also showed a broad immune response but with lower affinities than mAb 7G12. Moreover, mAb 6E7 was unable to block the biological activities of the different Tat variants, suggesting that this region was not reachable in active Tats.
MAb7G12 did not recognize any peptides covering the Tat Oyi sequence, and the affinities were comparable for all these heterologous Tats. Thus, we concluded that this antibody recognized sites on a common surface in all Tat variants. This common surface required a conserved folding of Tat variants that plays an important role for Tat activity. This conclusion is in disagreement with studies showing that Tat is an unfolded protein (41). The absence of recognition of all Tat variants after urea/heat degradation in ELISA and on Western blot analysis (data not shown) confirms the existence of a common 3D structure. The presence of a conserved folding in Tat variants is also supported by previous structural studies on three Tat variants with a biological activity (42). Accordingly, mAb 7G12 could be used to map the 3D epitope able to induce broadly cross-reactive antibodies. Preliminary recognition experiments with different Tat Oyi domains combinations have shown that the sequences 1-22, 38 -53, and 93-101 were involved in proper folding conditions to be recognized by mAb 7G12 (data not shown). A protection assay against acetylation on lysine residues could be an appropriate means to precisely map the discontinuous epitope but needs further development.
We showed that mAb 7G12 neutralized two biological activities for all Tat variants tested. We used a higher Tat concentration in the Jurkat cells apoptosis assay (5 M) compared with the transactivation assay with HeLa cells (0.5 M) because these two cell lines are differently sensitive to the extracellular Tat. For instance, Jurkat T-cells do not express caveolin and cannot internalize Tat by this pathway (43). Moreover, the full-length form of Tat is less efficient for apoptosis than the short form, whereas no difference is observed for the transactivation assay (44). In contrast, the quantity of antibodies used to obtain a high neutralization seemed less important in the apoptosis assay (20 g) than in the transactivation assay (50 g), but the final antibody concentrations were similar (0.2 g/l and 0.1 g/l, respectively). The lower Tat/antibody ratio needed to obtain complete neutralization in the transactivation assay arises from the efficiency of the LTR promoter. Thus, a molar:molar ratio (0.25 nmoles of Tat and 0.33 nmoles of mAb) is crucial to block all Tats able to trigger ␤-Gal expression. In contrast, trigger apoptosis pathways are more difficult, especially in a stable cell line, and the neutralization is easier, needing a higher Tat:antibody ratio (0.65 nmoles of Tat and 0.14 nmoles of mAb). We observed a low neutralization of Tats Oyi and HxB2 transactivation activities with mAb 27A8 (Fig. 6A). This antibody recognized a common linear epitope in the C-terminal domain of these two clade B variants. mAb 27A8 could interfere with their transactivation activities but with a very low efficiency com- FIGURE 5. mAb 7G12 blocks Tat uptake. Tat uptake into Jurkat cells was analyzed in the absence or presence of mAb 7G12 or 27A8 by anti-Tat immunoblotting on nuclear and cytosolic fractions. A, Western blotting was performed with the indicated antibodies. Annexin I and P-AC-histone H3 antibodies were used as control of cytosolic and nuclear fractions, respectively. This blot is representative of three independent experiments. Fractionation efficiency was controlled in the supplemental data with these antibodies and with a membrane-specific antibody (Fusin). B, percentage of translocated Tat (mean Ϯ S.D., n ϭ 3) was analyzed by densitometry imaging. The sum of densitometries in nuclear and cytosolic fractions without antibodies (control) represents 100% of translocated Tat in each experiment. Total was the sum of percentages obtained in nuclear and cytosolic fractions. *, p Ͻ 0.05 between percentages total in the absence or presence of antibodies; **, p Ͻ 0.05 between percentages in the absence or presence of antibodies in each compartment.
pared with mAb 7G12. Moreover, mAb 27A8 was unable to prevent apoptosis activities of these Tats.
Intracellular pathways leading to Tat induced transactivation and apoptosis were probably distinct but both dependent on Tat uptake. We observed that mAb 7G12 was able to block Tat uptake in both cell lines. Kinetic conditions used resulted from data published previously (44), highlighting that internalization was almost completed after 2 h. Experimental conditions were close to those of the transactivation assay but with 5 M (and not 0.5 M) of Tat. A highest Tat concentration was used to obtain a good signal in nuclear and cytosolic fractions on immunoblot analysis. However, because of experimental limitations, a molar:molar ratio between Tat and mAb was not reached in these conditions, explaining why 19% of Tat was still observed in the nucleus (Fig. 5). We assume that, with a molar: molar ratio, mAb 7G12 could completely block Tat uptake. mAb 27A8 had a low effect on Tat Oyi uptake, it was not significant in the assay. In molar:molar conditions, this effect could become significant, as observed in figure 6A, on transactivation activities of the Oyi and HXB2 Tat variants. However, mAb 27A8 was inefficient compared with mAb 7G12 and did not inhibit the other variants without the specific C-terminal linear epitope.
Up to now, only antiretroviral therapy (ART) is an efficient treatment against HIV-1 but does not eradicate the virus (45). Stopping treatment triggers the viral production because of the existence of reservoir cells (46). Passive immunization with a neutralizing antibody targeting other pathways would complement ART. Thus, mAb 7G12 properties are interesting for this purpose after humanization. We showed that Tat is a naturally folded protein in the blood of HIV-infected patients and can generate antibodies against 3D epitopes (15). Thus, therapeutic antibodies recognizing 3D epitopes of Tat could be selected by competitive ELISA with mAb 7G12 on isolated B-cells from long-term non-progressor patients. Studies also suggested that mAbs combination might protect more effectively against HIV-1 (47). Thus, passive immunization, using broadly neutralizing antibodies against different targets, such as Tat and gp120, should be evaluated for therapeutic potential.