1 E03-34 INSERM, Institut de Génétique Moléculaire, Hôpital St Louis, 27 rue Juliette Dodu, 75010 Paris, France
2 UPR9021 CNRS, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France
3 Centre de Recherches des Cordeliers, Université Pierre et Marie Curie, 75005 Paris, France
Correspondence
C. Desgranges
claude.desgranges{at}chu-stlouis.fr
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ABSTRACT |
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Present address: UPR 2228 CNRS, 45 rue des Saint-Pères, 75006 Paris, France.
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INTRODUCTION |
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Anti-Tat antibodies are present in the sera of HIV-1-infected patients (Reiss et al., 1991; Rodman et al., 1993
). Epidemiological studies have shown that high titres of anti-Tat antibodies correlate with the maintenance of long-term non-progression status in the course of HIV-1 infection (Re et al., 2001
; Richardson et al., 2003
; Zagury et al., 1998
). Natural IgM antibodies reacting with Tat may influence the course of AIDS progression and provide an early initial defence against the pathological effects of the Tat protein after HIV infection (Rodman et al., 1993
, 2001
). In vitro, anti-Tat antibodies abrogate Tat-dependent TNF-
secretion by monocytes (Bennasser et al., 2002
), inhibit Tat-induced Kaposi's sarcoma cell proliferation (Ensoli et al., 1990
) and block Tat transactivation of the HIV-1 LTR (Brake et al., 1990b
). Furthermore, monoclonal antibodies interfering with extracellular Tat modulate HIV-1 replication in infected cell cultures (Moreau et al., 2004; Re et al., 1995
; Steinaa et al., 1994
), suggesting that Tat may be a possible target for specific immunotherapy in HIV-1-infected patients. Immunization with a chemically inactivated Tat toxoid elicits high titres of anti-Tat antibodies in the serum of human volunteers (Gringeri et al., 1999
). Two immunodominant human B-cell epitopes with very limited antigenic polymorphism have been identified in the N-terminal (aa 120) and basic (aa 4461) regions of the Tat protein (Noonan et al., 2003
). Vaccination with short synthetic peptides covering these epitopes (aa 416 and 5366) induced only moderate protection after virus challenge in macaques (Goldstein et al., 2000
). However, encouraging results have been obtained in animals vaccinated with the active Tat protein (Cafaro et al., 1999
) or its toxoid form (Pauza et al., 2000
), although no protection was observed against challenge with a highly pathogenic strain of simian/HIV (Silvera et al., 2002
).
In this study, we analysed the antibody response against Tat in the sera of rabbits, monkeys, mice and humans immunized with the full-length recombinant Tat protein, synthetic Tat, Tat toxoid or Tat synthetic peptides. The capacity of these serum antibodies to block extracellular Tat transactivation and to reduce virus replication in infected cell cultures was analysed with regard to their fine specificity.
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METHODS |
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The 853 and 1953 Tat fragments were synthesized by the solid-phase method as described previously (Cornille et al., 1999). Peptides 120 and 4461 were synthesized using classical Fmoc solid-phase chemistry (Belliard et al., 2003
; Neimark & Briand, 1993
). A complete set of HIV-1 clade B consensus short overlapping Tat peptides (15 aa) was obtained through the AIDS Research and Reference Reagents Program (Division of AIDS, NIAID, NIH, Bethesda, MA, USA).
Sera.
Two mouse sera (Mur1 and Mur2) were obtained from BALB/c mice immunized by the intranasal route with sTat in the presence of double-stranded RNA motifs as an adjuvant (C. D. Partidos, J. Hoebeke, E. Moreau, M. Tunis, G. Belliard, J. P. Briand, C. Desgranges & S. Muller, unpublished data). Three Tat-specific rabbit serum samples were obtained from animals immunized as described previously (Belliard et al., 2003). Rabbit 1 (Rab1) was immunized with Tat peptide 853, rabbit 2 (Rab2) with Tat peptide 1953 and rabbit 3 (Rab3) with Tat peptide 4461. Peptides were injected subcutaneously with complete Freund's adjuvant. Antiserum from rabbit 4 (Rab4) was raised against a recombinant HIV-1 Tat protein and obtained through the AIDS Research and Reference Reagents Program (Hauber et al., 1987
). Two macaque rhesus monkeys (Mac1 and Mac2) were immunized intramuscularly with a mixture of Tat peptides 120, 161 and 4461 (Belliard et al., 2003
). Mac3 antiserum (a gift from B. Verrier and M. Girard, ENSL, Lyon, France) was obtained from a macaque injected with rTat. Two human sera were obtained from HIV-negative volunteers (Hum1 and Hum2) vaccinated intramuscularly with Tat toxoid (Gringeri et al., 1999
).
ELISA.
The reactivity of antisera with Tat peptides and Tat protein was determined by ELISA. Briefly, 100 ng rTat or Tat peptides in 0·05 M carbonate buffer, pH 9·6, was coated on to 96-well MaxiSorp (Nunc) microtitre plates for rTat or Immunolon 4 HBX (Dynex) plates for the peptides at 37 °C overnight. Wells were blocked with PBS containing 3 % (w/v) BSA at 37 °C for 2 h. One hundred microlitres of serial serum dilutions were then added to each well and incubated at 37 °C for 2 h. Wells were washed three times with PBS containing 0·1 % (v/v) Tween 20 (PBS/T) followed by incubation with 100 µl horseradish peroxidase (HRP)-labelled secondary antibody at 37 °C for 1 h. HRP-labelled antibodies were from Dakopatts for the detection of human and rabbit IgG and from Jackson ImmunoResearch Laboratories for the detection of mouse and monkey IgG. After three washes with PBS/T, the assay was developed with citrate buffer (40 mM citric acid, 100 mM Na2HPO4, pH 7) containing 0·15 mg o-phenylenediamine ml1 and 0·1 % (v/v) H2O2. The reaction was stopped with 20 µl 1 M H2SO4 and the absorbance values measured at 492 nm. The cut-off values for each assay were determined with pre-immune sera (macaques) or with sera from non-immunized animals (mice, rabbits) and humans. Test sera were considered positive when the absorbance values were higher than the mean A492+2 SD of non-immune controls.
SDS-PAGE and Western blot analysis.
SDS-PAGE was carried out as described by Laemmli (Laemmli, 1970) with 10 µg rTat run on a 15 % gel and electrophoretically transferred to PVDF membrane (Hybond-P; Amersham Pharmacia) by standard procedures. rTat was detected using the different antisera and the appropriate HRP-conjugated secondary antibody as described above and visualized by enhanced chemiluminescence Western blotting reagent (ECL+; Amersham Biosciences).
Transactivation assay.
Transactivation was assayed by inducing Tat-dependent chloramphenicol acetyltransferase (CAT) production in HeLa cells harbouring an integrated HIV-1 LTRCAT gene construct (HL3T1 cells) (Felber & Pavlakis, 1988). HL3T1 cells were seeded into a 24-well plate (4x104 cells per well) and incubated in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10 % fetal calf serum (FCS) overnight. To assay the transactivation ability of Tat preparations, rTat and sTat were dissolved in DMEM/10 % FCS (final volume 500 µl) supplemented with 100 µM chloroquine (Sigma) and added to the cells. After 24 h, cells were washed twice with PBS and incubated for an additional 24 h in fresh DMEM/10 % FCS. Cells were lysed and CAT production was assayed with a CAT ELISA kit (Roche Diagnostics) following the manufacturer's instructions. To test the anti-Tat antibody neutralizing properties, rTat protein was diluted in DMEM/10 % FCS (final volume 500 µl) supplemented with 100 µM chloroquine to a final concentration of 25 ng ml1 and incubated with various dilutions of immune serum at 37 °C for 1 h. The mixture was then added to the cells and the procedure carried out as described above.
Inhibition of virus replication.
H9 cells (1x106) were infected with the HIV-1 IIIB laboratory strain (p24 concentration of 200 ng ml1) at 37 °C in 5 % CO2 overnight. Cells were washed twice in culture medium and seeded (4x104 cells per well) in 96-well plates with or without different dilutions of antisera. The cultures were incubated at 37 °C in 5 % CO2 for 5 days. Aliquots of culture supernatants were sampled daily and replaced by fresh medium containing the respective concentrations of antibodies. Virus replication was monitored by quantification of HIV-1 p24 core antigen using a capture ELISA as described previously (Cartier et al., 1999).
Anti-Tat antibodies were added to long-term chronically HIV-1 IIIB-infected H9 cells with or without rTat protein (100 ng ml1) for 4 or 6 days and the p24 concentration measured as described above.
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RESULTS |
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Antisera from macaques Mac1 and Mac2 immunized with a mixture of Tat peptides (120, 4461 and 161) displayed high antibody specificity to the N-terminal region (titres=51 200 and 102 400, respectively). Mac1 also mounted a high antibody response to the basic peptide 4461, whereas Mac2 produced an equivalent response to peptides from the core (peptides 1953 and 3751) and basic regions (peptide 4461). Serum raised against rTat in Mac3 displayed antibody specificity to the N-terminal, core and basic regions, although antibody titres were weaker than those measured in the other two macaques. All macaque antisera recognized rTat in both ELISA and Western blotting (Fig. 1c) but showed a more limited reactivity to sTat in ELISA.
The two Hum1 and Hum2 sera collected from individuals immunized with Tat toxoid displayed a broad range of antibody specificities. In particular, they recognized peptides 120, 853 and 4461, covering the N-terminal and the basic regions of Tat, and also the C-terminal RGD motif present in residues 7779 not seen with the other antisera. Antibodies from both Hum1 and Hum2 sera bound rTat and sTat proteins in ELISA, and in Western blotting stained a band that corresponded to rTat protein (Fig. 1d).
Inhibition of extracellular Tat LTR transactivation
Exogenous Tat can enter HL3T1 cells harbouring an LTRCAT plasmid and transactivate CAT production. Both rTat and sTat were active in this assay and displayed significant transactivation properties in the same range of concentration (data not shown). However, due to limited availability of material, the ability of Tat antibodies to interfere with transactivation was tested with rTat only. Dilutions of Tat antisera were pre-incubated with rTat before addition to the reporter cell line. Pre-immune sera used as controls did not exhibit significant inhibition of CAT production and were considered as a negative control for each experiment. In contrast, antibodies from mouse Mur1 diluted 1 : 100 were able to block more than 40 % of Tat transactivation and this inhibition was retained at a 1 : 200 serum dilution. Mur2 antibodies did not neutralize Tat activity (Fig. 2a). It was noticeable that the only difference between these two antisera was their reactivity with the basic region of Tat (Tables 1 and 2
).
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Among the three monkey antisera tested, Mac1 antibodies displayed the strongest inhibition of Tat transactivation (>80 % at a 1 : 100 dilution), which was retained at a 1 : 200 serum dilution (Fig. 2c). Antibodies from Mac2 and Mac3 also inhibited Tat activity but this effect was diminished when the serum was further diluted. It is important to note that all these antisera reacted with rTat protein and with the N-terminal and basic regions of Tat. In this group of antisera, Mac1 antibodies bound peptide 4461 with the highest titre (204 800).
The two Hum1 and Hum2 sera from Tat toxoid-vaccinated subjects contained antibodies with a high potential to inhibit Tat transactivation (Fig. 2d). At a 1 : 200 dilution, both immune sera inhibited more than 80 % of the control transactivation and 40 and 50 % inhibition was still detectable at a 1 : 1000 for Hum1 and Hum2, respectively (Fig. 2d
). These two sera were also positive with the rTat protein and with the N-terminal and basic regions.
Inhibition of virus replication
After 4 days, newly infected H9 cells produced 625 ng HIV-1 p24 core antigen ml1 in culture supernatants. As shown in Fig. 3, mouse Mur1 and Mur2 antibodies did not display any visible effect against virus replication. Also, antibodies from rabbits Rab1, Rab2 and Rab3 failed to inhibit HIV-1 replication, whereas antibodies from rabbit Rab4 exerted a 25 % inhibition of p24 production. Antibodies from Mac1 and Mac2 monkeys blocked 40 and 25 % of virus replication, respectively, whereas Mac3 antibodies did not show any significant inhibition of p24 production. In contrast, a 1 : 20 dilution of Hum1 serum inhibited up to 75 % of virus replication. No inhibition was observed with a 1 : 100 dilution of any of the animal and human sera (data not shown). Hum2 antibodies were not tested in this assay as our serum sample was exhausted in previous studies.
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DISCUSSION |
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Previous studies have reported that the Tat N terminus and basic regions contain immunodominant epitopes in mice (Boykins et al., 2000), rabbits (Goldstein et al., 2001
), macaques (Tikhonov et al., 2003
) and humans (Re et al., 2001
; Tahtinen et al., 1997
). In the present study, we first confirmed that these two regions contained dominant B-cell epitopes recognized by IgG antibodies present in sera from Mur1, Rab4, Mac3, Hum1 and Hum2 raised against the full-length 86 aa protein, administered either as rTat, sTat or Tat toxoid. Antibodies from two monkeys immunized with a cocktail of synthetic peptides (120, 161 and 4461) reacted equally well with peptides 120 and 4461, though the antibody titre to peptide 4461 was eightfold higher in Mac1 than in Mac2. With the exception of the antiserum Rab3 raised against peptide 4461, all sera examined in our study reacted with rTat and sTat in ELISA and/or with rTat in its denatured form in Western blotting. However, Rab4 antibody titre to sTat was significantly lower than that measured for the rTat protein. In addition, Rab4 serum reacted with all long Tat fragments but only with the 115 short peptide. Epitope recognition by Rab4 antibodies could be highly dependent on the folding of the Tat protein preparation. Rab2 antibodies bound weakly to rTat and sTat in ELISA but reacted with denatured rTat, suggesting that the epitopes recognized by Rab2 antibodies in the core region are probably buried within the folded Tat structure and exposed when the protein is tested in denaturing conditions. Rab3 serum raised against the basic region peptide 4461 reacted with immunizing peptide but not with the other large Tat fragments (853, 1953) or overlapping short peptides in any assay. Rab3 antibody reactivity was low with rTat in ELISA but dramatically increased when sTat was used as an antigen. It has been shown previously that the binding of antibodies to the basic region of Tat is highly dependent on protein folding (Moreau et al., 2004
; Tosi et al., 2000
). Moreover, amino acid residues flanking the basic region can hinder the binding of specific antibodies to the minimal antigenic sequence (Goldstein et al., 2001
). It is likely that the epitope structure as presented by sTat is similar to the folding of the peptide 4461 used for immunization. This particular structure could be absent in overlapping peptides or in rTat in its active form or under the denaturing conditions used in Western blotting.
The ability of antisera to neutralize exogenous Tat transactivation was assessed by an LTRCAT assay. It is well established that monoclonal or polyclonal antibodies directed against the various epitopes of the Tat protein can abolish LTR transactivation induced by exogenous Tat (Tikhonov et al., 2003; Tosi et al., 2000
). Our results extend these findings by demonstrating that only immune sera with antibody responses to both N-terminal and basic regions (namely Mur1, Rab4, Mac1, Hum1 and Hum2) were able to block extracellular transactivation significantly. Moreover, it seems that the extent of their neutralizing activity correlated with responses toward the basic domain of Tat. Thus, in the case of Mac2 and Mac3, sera that had low IgG titres to peptide 4461 displayed a weak neutralizing potential against Tat compared with Mac1 serum. However, it was noticeable that Hum1 and Hum2 sera displayed the highest neutralizing ability, although they had moderate antibody titres against both N-terminal and basic regions. It is well known that the neutralizing potential of serum antibodies to a given antigen does not systematically correlate with antibody titres measured in ELISA (Alape-Giron et al., 1997
; Simonsen et al., 1987
) but to the antibody affinity for the target antigen (Olszewska et al., 2000
). Hum1 and Hum2 antibodies are likely to display a high affinity for rTat protein.
Neutralization of extracellularly secreted Tat by antibodies leads to partial inhibition of virus replication in cultures of T-cell lines newly infected with HIV-1, but requires high concentrations (µg ml1) of monoclonal antibodies in culture medium (Moreau et al., 2004; Re et al., 1995
; Steinaa et al., 1994
). The Hum1 serum that exhibited this activity may have been the only one that contained enough specific antibodies to abrogate Tat autocrine/paracrine activity significantly.
In chronically infected cells producing stable low levels of p24 antigen, the presence of Hum1 antibodies did not reduce virus production. This may be due to lower expression of extracellular Tat in these cells compared with Tat production in acutely infected cells (Ensoli et al., 1993). However, addition of soluble active Tat protein to chronically infected cells enhanced virus replication, as previously demonstrated by Boykins et al. (2000)
, and this activation was totally inhibited by Hum1 antibodies. These results suggest that anti-Tat antibodies in vivo could block the Tat protein secreted by HIV-productive cells that could reactivate virus replication in neighbouring chronically infected cells.
In conclusion, the data presented in this study demonstrate that immunization with the whole 86 aa Tat protein or a multi-epitope peptide cocktail leads to the production of Tat-specific antibodies able to neutralize exogenous Tat-driven transactivation and disrupt the autocrine/paracrine effects of the protein involved in virus replication in vitro. Therefore, with regard to the pleiotropic effects of extracellular Tat in the course of HIV-1 infection, our findings lend weight to the conclusion that active or passive Tat immunization could be used advantageously as a support therapy in association with other antiretroviral drugs.
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ACKNOWLEDGEMENTS |
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Received 15 June 2004;
accepted 28 June 2004.
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