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Antibodies against human IFN-α and -β recognized the immunosuppressive domain of HIV-1 gp41 and inhibit gp41-binding to the putative cellular receptor protein p45
Immunology Letters 69 (1999) 253 – 257
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Antibodies against human IFN-a and -b recognized the immunosuppressive domain of HIV-1 gp41 and inhibit gp41-binding to the putative cellular receptor protein p45 Ying-Hua Chen a,*, Weicheng Wu a, Jun Yang b, Sen-Fang Sui b, Junfeng Sun a, Manfred P. Dierich c a
Department of Biological Science and Biotechnology, Laboratory of Immunology, Tsinghua Uni6ersity, Beijing 100084, PR China b Department of Biological Science and Biotechnology, Biophysics Laboratory, Tsinghua Uni6ersity, Beijing 100084, PR China c Institute of Hygiene, Uni6ersity of Innsbruck, Ludwig-Boltzmann-Institute for AIDS-Research, A-6010 Innsbruck, Austria Received 12 January 1999; accepted 25 March 1999
Abstract Sequence-comparison indicates existing sequence-similarity between receptor-binding regions of human type 1 IFNs (IFN-a, -b and -v) and HIV-1 gp41. Previous findings had suggested that the increased levels of antibodies against human IFN-a and -b in HIV-1-infected individuals are associated with a common epitope on gp41, IFN-a and -b. To clarify the relationship between human type I interferon and HIV-1 gp41 and the protective mechanism of an IFN-a-vaccine, we prepared antisera against human IFN-a, -b and HIV-1 gp41, and examined crossreaction of these antisera and their inhibition of gp41 binding to its binding protein p45. Mouse antisera against IFN-a and -b could recognize HIV-1 recombinant soluble (aa539 – 684) and gp41 immunosuppressive peptide (ISP, aa583–599), while normal mouse sera (pre-immune sera) did not. Mouse antisera to rsgp41 crossreacted with IFN-a and -b. Besides, mouse antisera to IFN-a and b, like mouse anti-rsgp41 antiserum, could inhibit gp41-binding to its putative cellular receptor protein p45, while normal mouse serum did not. These results indicate that antibodies crossreacting with gp41 ISP, IFN-a and -b, could be induced by this common immunological epitope in vivo. © 1999 Elsevier Science B.V. All rights reserved. Keywords: HIV-1 gp41; IFN-a; IFN-b; Putative receptor protein
1. Introduction The human immunodeficiency virus type 1 (HIV-1) after binding by the envelope protein gp120 to its cellular receptor CD4 and coreceptor CCR-5 or CXCR4 infects susceptible cells [1]. The binding of gp120 with CD4 and CCR5 or CXCR4 induces significant and rapid conformational changes of gp41. It results in increased exposure of gp41 and ultimately leads to the dissociation of gp120 [2]. The C-terminal peptide of the heptad repeat binds to the N-terminal peptide coiled coil and induces the fusion-active conformation [3]. Recent studies including inhibition of cell fusion by antibodies [4], peptide [5] and mutation of * Corresponding author. Fax: +86-10-62785505. E-mail address: [email protected] (Y.-H. Chen)
different sites in gp41 have indicated that HIV-1 gp41 plays an important role in virus uptake. From these studies two regions, namely aa583–599 (part of the N-terminal peptide) and aa645–675 (C-terminal peptide), in addition to the N-terminal aa517–527 have turned out to be of special importance for cell fusion and viral uptake. Antibodies against regions in SIV gp32 homologous to both regions in HIV-1 gp41 could protect macaques from SIV-infection [6]. The first region (aa583–599) in gp41 was demonstrated as an immunosuppressive domain (ISD). It was demonstrated by us, and Henderson’s group that gp41 by ISD could bind to a putative cellular receptor protein p45 (45 kDa) on the cell surface of human T, B and monocytic cells, and p45 was isolated from cell lysates of these cell lines by affinity-chromatography using rsgp41 or immunosuppressive peptide (Env aa583–599)-sepharose column [7–9].
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Y.-H. Chen et al. / Immunology Letters 69 (1999) 253–257
The role of interferon-a and -b (IFN-a and -b in HIV-infection is complex and only incompletely understood. Administration of IFN-a to AIDS-patients does not prevent progression of the disease [10]. A 2-year study of an anti-IFN-a immunization of HIV-infected patients has demonstrated that IFN-a-vaccine-treated HIV-infected patients showed a significant delay in disease progression which was associated with an antiIFN-a antibody response and increase of its IFN-aneutralizing capacity [11,12]. We found that levels of antibodies against human IFN-a and -b in HIV-1-infected individuals were increased [13,14], and sequencesimilarity exists between human type I interferon (IFN-a, -b and -v) and HIV-1 gp41 [15]. Previous studies suggested that increased levels of antibodies against human IFN-a and -b in HIV-1-infected individuals may be associated with a common immunological epitope [13,14]. In this study, we wanted to induce antibodies against human IFN-a, -b and HIV-1 gp41 in vivo, and to examine cross-reaction of these antibodies and inhibition of gp4 1 binding to its putative cellular receptor protein p45.
weeks old) or from mice immunized using human IFNa or IFN-b (1× l06 U/immunization) with Freunds adjuvant for four times. Peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (P260) were obtained from Dako (Denmark). The putative cellular receptor protein P45 was isolated from Raji cell lysates by affinity-chromatography using rsgp41column (described in [16]).
2. Materials and methods
2.4. Inhibition of gp41 -binding to P45 in SPR-assay
2.1. HIV-1 gp41 protein and peptides
Surface plasmon resonance (SPR) measurement had been used for measuring molecular interactions [17]. By a SPR-instrument we measured specific binding of biotin–avidin [18]. The mechanism and procedure of measuring molecular interactions were described in [13,14]. Briefly a HL6711G semiconductor laser (LD) was used as the source of p-polarized monochromatic light of 670 nm. The intensity of the light reflected was monitored by a silicon photodiode. A turbo C program was used to collect data and transfer them to a computer in the form of reflectance as a function of real time. The triangular prism (nD = 1.8) and a sample cell (200 m1) was positioned on a small rotary table. Metal coatings of gold (50 nm thick) were prepared by evaporation on glass slides. The gold surfaces were coated polystyrene. The slides were then struck to the prism by an index-matching oil, whose refractive index (nD) was 1.516. The interval of measurements was 100 s. In order to examine the inhibition of interaction between rsgp41 and the putative receptor protein p45 by antisera to IFN-a, -b and rsgp41, measurements were made as follows: after equilibrium by 0.1 M NaHCO3 (pH 9.6), rsgp41 (4 mg) was coated to slide. Then the sample cell was washed by PBS, and antisera or normal serum (pre-immune serum as negative control), were added. After washing by PBS, the putative receptor protein p45 was pumped in to sample cell. Finally, the sample cell was washed by PBS. Data were collected and transferred to a computer in the form of reflectance as a function of real time.
The recombinant soluble gp41 (rsgp41) from Biotest (Dreieich, Germany), represents the external portion of the transmembrane protein gp41 of HIV-1 (derived from clone BH10) [7]. The amino acid sequence of rsgp41 (Env aa539– 684): AR SMTLTVQARQ LLSGIVQQQN NLLRAIEAQQ HLLQLTVWGI KQLQARILAV ERYLKDQQLL GIWGCSGKLI CTTAWPWNAS WSNKSLEQIW NNMTWMEWDR EINNYTSLIH SLIEESQNQQ EKNEQELLEL DKWASLWNWF NITN. Rsgp41 was expressed in Escherichia coli. After purification, rsgp41 was resuspended in 0.1 mol/1 Tris – HCl (pH 9.0) supplemented with 0.2% sodium dodecyl sulphate (SDS), 0.3 mol/l urea and 10% glycerol. The protein migrates on SDSpolyacrylamide gel electrophoresis (PAGE) (0.1% SDS) as a single band, with molecular weight of 1 8 kD. ISP (immunosuppressive peptide) was synthesized according to the sequence of the HIV-1 isolate IIIB: HIV Env aaS83–599, LQARILAVERYLKDQQL [7,9].
2.2. Interferons, antibodies and protein P45 Recombinant human IFN-a was kindly provided by Bender (Vienna, Austria). Recombinant human IFN-b was obtained from Dr Rentschler Biotechnologie GmbH (Laupheim, Germany). The normal mouse sera and mouse antisera to human IFN-a and -b were obtained from healthy pre-immune Balb/c mice (6
2.3. Enzyme-linked immunosorbent assay (ELISA) Rsgp41 and peptide (10 mg/ml) or interferons (5× 105 U/ml) were coated overnight on a microtiter plate at 4°C. Nonspecific binding was blocked by incubation with 1% BSA or 0.3% gelatine in PBS. After washing three times with PBS–Tween 20 (0.1% Tween 20), mouse antisera (in dilution 1:20 in PBS) and pre-immune sera were added and incubated for 1 h at room temperature. The plate was washed again with PBS– Tween 20. Peroxidase-conjugated rabbit anti-mouse antibody were added. After further washing, freshly prepared 2,2%-azino-di-(3-ethylbenzthiazoline sulfonate)peroxide solution was added and the optical density was measured.
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Fig. 1. Mouse anti-human IFN-a and -b antisera recognized rsgp41 and ISP in ELISA-assay. Fig. 1a show crossreaction of anti-IFN-a and -b antisera (lane A – H) and show control sera (normal mouse sera) (lane I – L) with rsgp41; Fig. 1b show cross-reaction of anti-IFN-a and -b antisera (lane A – D) and control serum (lane E and F) with ISP. Coating of antigen: rsgp41 (2 mg/ml) (Fig. 1a); ISP (4 mg/ml) (Fig. 1b). AS-alpha-1 and AS-alpha-2: anti-IFN-a antisera from two mice (1:20 and 1:100 dilutions). AS-beta-1 and AS-beta-2: anti-IFN-b antisera from two mice (1:20 and 1:100 dilutions). NMS, normal mouse serum (1:20 and 1:100 dilutions). The figure shows data from three separate experiments.
3. Results and discussion Recently we have reported that a common epitope exists between IFN-a, -b and HIV-1 gp41 [15], and the common epitope was located in the receptor-binding regions of gp41 and of human type I interferon, namely, in the immunosuppressive domain (ISD, aa583–599) of gp41 and in two regions (aa29 – 35 and 123 – 140) of human IFN-a, -b and -v, which form IFN-a/b-receptor binding domains [15]. To clarify the functional mechanism of IFN-a-vaccine and relationship between human type I interferon and HIV-1 gp41, we prepared antibodies against IFN-a, -b and gp41, and examined crossreaction of these antibodies. Mouse antisera to IFN-a and -b could recognize rsgp41 (aa539–684) (Fig. 1a, A – H), while two normal mouse sera (pre-immune sera) did not, and showed only a background (Fig. 1a, I – L). Moreover, the antisera could recognize immunosuppressive peptide (aa583– 599) of gp41 (Fig. 1b, A – D). Besides, mouse antiserum against rsgp41 could bind human IFN-a and -b (Fig. 2). These results indicate that the common immunological epitope exists in human IFN-a, -b and gp41 ISP, suggesting that the antibodies induced in HIV-infected patients by IFN-a-vaccine could bind gp41 ISP. In fact, Gringeri had observed that HIV-infected patients treated with a IFN-a-vaccine showed increase of significant antibody titer against gp41 peptide aa560 – 599 and a significant reduction of disease progression [11], providing an important clinical evidence for our findings. Based on the results that antisera to human IFN-a and -b crossreacted with rsgp41 (Fig. 1a) and ISP (Fig. 1b), we examined whether the antisera to human IFN-a and -b like anti-rsgp41 antiserum inhibit binding of the putative receptor protein P45 to rsgp41 in SPR-assay.
Rsgp41 was coated to slide (Fig. 3, line A–B). Then the sample cell was washed by PBS (Fig. 3, line B–C), and normal mouse serum (NMS) (Fig. 3a, b, c, line C–D) or gelatine (Fig. 3d, line C–D) were added to block nonspecific binding. After washing by PBS (Fig. 3, line D–E), the putative receptor protein p45 was pumped into the sample cell. The line F–G shows the case after washing by PBS. The result indicates clearly that rsgp41 interacts with the putative receptor protein P45 (Fig. 3, line E–F). In the inhibition experiments, after addition of mouse antisera to human IFN-a and b and rsgp41 (instead of normal mouse serum or gelatine), the adsorption (Fig. 3a, line C1 –D1; Fig. 3b, C2 –D2; Fig. 3c, C3 –D3) showed stronger then the adsorption the ease of normal mouse serum (Fig. 3a, b, c, line C–D),and the protein p45 did not adsorb (Fig. 3a, line E1 –F1;
Fig. 2. Mouse anti-gp41 antiserum recognized IFN-a and -b in ELISA-assay. NMS, normal mouse serum; AS, mouse anti-rsgp41 serum. Coating of antigen: IFN-a (2 mg/ml); IFN-b (2 mg/ml). The figure shows data from two separate experiments.
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Fig. 3. Inhibition of gp41-binding to the putative receptor protein p45 by blocking the receptor binding site (aa583 – 599) of gp41 using antisera to human IFN-a (Fig. 3a), -b (Fig. 3b) and rsgp41 (Fig. 3c) in SPR-assay. NMS, normal mouse serum. AMS-IFN-a, mouse antiserum to IFN-a. AMS-IFN-b, mouse antiserum to IFN-b. AMS-gp41, mouse antiserum to rsgp41. The lines noted absorptions of proteins between 0 and 260 min. Lines A – B, A1 – B1, A2 – B2 and A3 –B3, absorption of gp41 to slide. Lines B – C, B1 – C1, B2 – C2 and B3 – C3, adsorption after washing using PBS. C–D, normal mouse serum (pre-immune serum); C1 –D1, adsorption of antiserum to IFN-a; C2 – D2: adsorption of antiserum to IFN-b; C3 –D3, adsorption of antiserum to rsgp41. Lines D–E, D1 –E1, D2 –E2 and D3 – E3, adsorption after washing using PBS. Lines E – F, E1 – F1, E2 –F2 and E3 –F3, adsorption of p45. Lines F–G, F1 –G1, F2 –G2 and F3 – G3, adsorption after washing using PBS. Fig. 3d shows p45-binding to rsgp41 by using gelatine to block nonspecific binding.
Fig. 3b, E2 –F2; Fig. 3c, E3 – F3), suggesting that antisera to human IFN-a, -b and rsgp41 could inhibit binding of the putative receptor protein p45 to gp41. The inhibition of p45-binding to gp41 by the antisera to IFN-a and -b is due to blocking the receptor binding
site (ISD, aa583–599) on gp41, because the antiserums to IFN-a and -b could recognize gp41 ISP (aa583–599) (Fig. 1b). The result of this test suggests that the therapeutic effect of IFN-a-vaccine on HIV-infected patients may be associated with the inhibition of gp41-
Y.-H. Chen et al. / Immunology Letters 69 (1999) 253–257
binding to the putative cellular receptor protein p45 by the antibodies recognizing this common immunological epitope in the receptor binding region (immunosuppressive domain, aa583– 599) in gp41. Recent studies have demonstrated a critical step in the HIV entry mechanism that structural rearrangements in the gp41 core structure occur after gp120 binding to its receptors which induce exposure of the gp41 heptad repeat region [19,20], and binding of DP-178 peptide to the gp41 heptad repeat region can block the virus-dependent fusion [19,20], suggesting that the gp41 heptad repeat region (containing the common immunological epitope) is a critical structure for an effective vaccine.
Acknowledgements This work was supported by the 863-program, National Natural Science Foundation of China (Project NSFC-39770696 and NSFC-39880043), and by the China–Austria Cooperation (project V.B. 1).
References [1] G. Alkhatib, C. Combadiere, C.C. Broder, et al., Science 272 (1996) 1955 – 1958.
.
257
[2] P.L.St.J. Jones, T. Korte, et al., J. Biol. Chem. 273 (1998) 404 – 409. [3] W. Weissenhorn, A. Dessen, et al., Nature 387 (1997) 426–430. [4] S. Vanini, R. Longhi, A. Lazzarin, et al., AIDS 7 (1993) 167– 174. [5] S. Jiang, K. Lin, N. Strick, et al., Nature (Lond.) 365 (1993) 113. [6] M.G. Lewis, W.R. Elkins, F.E. McCutchan, et al., Vaccine 11 (1993) 1347 – 1355. [7] Y.H. Chen, C. Ebenbichler, R. Vornhagen, et al., AIDS 6 (1992) 533 – 539. [8] L.A. Henderson, M.N. Qureshi, J. Biol. Chem. 268 (1993) 15291 – 15297. [9] Y.H. Chen, M.P. Dierich, Immunol. Lett. 52 (1996) 153–156. [10] D. Zagury, I. Gagne, B. Reveil, et al., Lancet 2 (1985) 449. [11] A. Gringeri, E. Santagostino, P.M. Mannucci, et al., J. AIDS 7 (1994) 978 – 988. [12] D. Zagury, J. Bernard, A. Halbreich, et al., J. AIDS 5 (1992) 676 – 681. [13] Y.H. Chen, H. Stoiber, M.P. Dierich, Immunol. Lett. 55 (1997) 15 – 18. [14] Y.H. Chen, J.N. Feng, G. Stockl, et al., Molec. Immunol. 34 (1998) 1259 – 1262. [15] Y.H. Chen, M.P. Dierich, Immunol. Today 19 (1998) 586–587. [16] Y.H. Chen, G. Bock, R. Vornhagen, et al., Mol. Immunol. 31 (1994) 977 – 982. [17] M.C. Dubs, D. Altschuh, M.H.V. Van Regenmortel, Immunol. Lett. 31 (1991) 59 – 64. [18] Z. Liu, H. Qin, C. Xiao, et al., Eur. Biophys. J. 24 (1995) 31–38. [19] R.A. Furuta, C.T. Wild, Y. Weng, C.D. Weiss, Nature Stuct. Biol. 5 (1998) 276 – 279. [20] D.C. Chan, P.S. Kim, Cell 93 (1998) 681 – 684.
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Antibodies against human IFN-a and -b recognized the immunosuppressive domain of HIV-1 gp41 and inhibit gp41-binding to the putative cellular receptor protein p45 Ying-Hua Chen a,*, Weicheng Wu a, Jun Yang b, Sen-Fang Sui b, Junfeng Sun a, Manfred P. Dierich c a
Department of Biological Science and Biotechnology, Laboratory of Immunology, Tsinghua Uni6ersity, Beijing 100084, PR China b Department of Biological Science and Biotechnology, Biophysics Laboratory, Tsinghua Uni6ersity, Beijing 100084, PR China c Institute of Hygiene, Uni6ersity of Innsbruck, Ludwig-Boltzmann-Institute for AIDS-Research, A-6010 Innsbruck, Austria Received 12 January 1999; accepted 25 March 1999
Abstract Sequence-comparison indicates existing sequence-similarity between receptor-binding regions of human type 1 IFNs (IFN-a, -b and -v) and HIV-1 gp41. Previous findings had suggested that the increased levels of antibodies against human IFN-a and -b in HIV-1-infected individuals are associated with a common epitope on gp41, IFN-a and -b. To clarify the relationship between human type I interferon and HIV-1 gp41 and the protective mechanism of an IFN-a-vaccine, we prepared antisera against human IFN-a, -b and HIV-1 gp41, and examined crossreaction of these antisera and their inhibition of gp41 binding to its binding protein p45. Mouse antisera against IFN-a and -b could recognize HIV-1 recombinant soluble (aa539 – 684) and gp41 immunosuppressive peptide (ISP, aa583–599), while normal mouse sera (pre-immune sera) did not. Mouse antisera to rsgp41 crossreacted with IFN-a and -b. Besides, mouse antisera to IFN-a and b, like mouse anti-rsgp41 antiserum, could inhibit gp41-binding to its putative cellular receptor protein p45, while normal mouse serum did not. These results indicate that antibodies crossreacting with gp41 ISP, IFN-a and -b, could be induced by this common immunological epitope in vivo. © 1999 Elsevier Science B.V. All rights reserved. Keywords: HIV-1 gp41; IFN-a; IFN-b; Putative receptor protein
1. Introduction The human immunodeficiency virus type 1 (HIV-1) after binding by the envelope protein gp120 to its cellular receptor CD4 and coreceptor CCR-5 or CXCR4 infects susceptible cells [1]. The binding of gp120 with CD4 and CCR5 or CXCR4 induces significant and rapid conformational changes of gp41. It results in increased exposure of gp41 and ultimately leads to the dissociation of gp120 [2]. The C-terminal peptide of the heptad repeat binds to the N-terminal peptide coiled coil and induces the fusion-active conformation [3]. Recent studies including inhibition of cell fusion by antibodies [4], peptide [5] and mutation of * Corresponding author. Fax: +86-10-62785505. E-mail address: [email protected] (Y.-H. Chen)
different sites in gp41 have indicated that HIV-1 gp41 plays an important role in virus uptake. From these studies two regions, namely aa583–599 (part of the N-terminal peptide) and aa645–675 (C-terminal peptide), in addition to the N-terminal aa517–527 have turned out to be of special importance for cell fusion and viral uptake. Antibodies against regions in SIV gp32 homologous to both regions in HIV-1 gp41 could protect macaques from SIV-infection [6]. The first region (aa583–599) in gp41 was demonstrated as an immunosuppressive domain (ISD). It was demonstrated by us, and Henderson’s group that gp41 by ISD could bind to a putative cellular receptor protein p45 (45 kDa) on the cell surface of human T, B and monocytic cells, and p45 was isolated from cell lysates of these cell lines by affinity-chromatography using rsgp41 or immunosuppressive peptide (Env aa583–599)-sepharose column [7–9].
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Y.-H. Chen et al. / Immunology Letters 69 (1999) 253–257
The role of interferon-a and -b (IFN-a and -b in HIV-infection is complex and only incompletely understood. Administration of IFN-a to AIDS-patients does not prevent progression of the disease [10]. A 2-year study of an anti-IFN-a immunization of HIV-infected patients has demonstrated that IFN-a-vaccine-treated HIV-infected patients showed a significant delay in disease progression which was associated with an antiIFN-a antibody response and increase of its IFN-aneutralizing capacity [11,12]. We found that levels of antibodies against human IFN-a and -b in HIV-1-infected individuals were increased [13,14], and sequencesimilarity exists between human type I interferon (IFN-a, -b and -v) and HIV-1 gp41 [15]. Previous studies suggested that increased levels of antibodies against human IFN-a and -b in HIV-1-infected individuals may be associated with a common immunological epitope [13,14]. In this study, we wanted to induce antibodies against human IFN-a, -b and HIV-1 gp41 in vivo, and to examine cross-reaction of these antibodies and inhibition of gp4 1 binding to its putative cellular receptor protein p45.
weeks old) or from mice immunized using human IFNa or IFN-b (1× l06 U/immunization) with Freunds adjuvant for four times. Peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (P260) were obtained from Dako (Denmark). The putative cellular receptor protein P45 was isolated from Raji cell lysates by affinity-chromatography using rsgp41column (described in [16]).
2. Materials and methods
2.4. Inhibition of gp41 -binding to P45 in SPR-assay
2.1. HIV-1 gp41 protein and peptides
Surface plasmon resonance (SPR) measurement had been used for measuring molecular interactions [17]. By a SPR-instrument we measured specific binding of biotin–avidin [18]. The mechanism and procedure of measuring molecular interactions were described in [13,14]. Briefly a HL6711G semiconductor laser (LD) was used as the source of p-polarized monochromatic light of 670 nm. The intensity of the light reflected was monitored by a silicon photodiode. A turbo C program was used to collect data and transfer them to a computer in the form of reflectance as a function of real time. The triangular prism (nD = 1.8) and a sample cell (200 m1) was positioned on a small rotary table. Metal coatings of gold (50 nm thick) were prepared by evaporation on glass slides. The gold surfaces were coated polystyrene. The slides were then struck to the prism by an index-matching oil, whose refractive index (nD) was 1.516. The interval of measurements was 100 s. In order to examine the inhibition of interaction between rsgp41 and the putative receptor protein p45 by antisera to IFN-a, -b and rsgp41, measurements were made as follows: after equilibrium by 0.1 M NaHCO3 (pH 9.6), rsgp41 (4 mg) was coated to slide. Then the sample cell was washed by PBS, and antisera or normal serum (pre-immune serum as negative control), were added. After washing by PBS, the putative receptor protein p45 was pumped in to sample cell. Finally, the sample cell was washed by PBS. Data were collected and transferred to a computer in the form of reflectance as a function of real time.
The recombinant soluble gp41 (rsgp41) from Biotest (Dreieich, Germany), represents the external portion of the transmembrane protein gp41 of HIV-1 (derived from clone BH10) [7]. The amino acid sequence of rsgp41 (Env aa539– 684): AR SMTLTVQARQ LLSGIVQQQN NLLRAIEAQQ HLLQLTVWGI KQLQARILAV ERYLKDQQLL GIWGCSGKLI CTTAWPWNAS WSNKSLEQIW NNMTWMEWDR EINNYTSLIH SLIEESQNQQ EKNEQELLEL DKWASLWNWF NITN. Rsgp41 was expressed in Escherichia coli. After purification, rsgp41 was resuspended in 0.1 mol/1 Tris – HCl (pH 9.0) supplemented with 0.2% sodium dodecyl sulphate (SDS), 0.3 mol/l urea and 10% glycerol. The protein migrates on SDSpolyacrylamide gel electrophoresis (PAGE) (0.1% SDS) as a single band, with molecular weight of 1 8 kD. ISP (immunosuppressive peptide) was synthesized according to the sequence of the HIV-1 isolate IIIB: HIV Env aaS83–599, LQARILAVERYLKDQQL [7,9].
2.2. Interferons, antibodies and protein P45 Recombinant human IFN-a was kindly provided by Bender (Vienna, Austria). Recombinant human IFN-b was obtained from Dr Rentschler Biotechnologie GmbH (Laupheim, Germany). The normal mouse sera and mouse antisera to human IFN-a and -b were obtained from healthy pre-immune Balb/c mice (6
2.3. Enzyme-linked immunosorbent assay (ELISA) Rsgp41 and peptide (10 mg/ml) or interferons (5× 105 U/ml) were coated overnight on a microtiter plate at 4°C. Nonspecific binding was blocked by incubation with 1% BSA or 0.3% gelatine in PBS. After washing three times with PBS–Tween 20 (0.1% Tween 20), mouse antisera (in dilution 1:20 in PBS) and pre-immune sera were added and incubated for 1 h at room temperature. The plate was washed again with PBS– Tween 20. Peroxidase-conjugated rabbit anti-mouse antibody were added. After further washing, freshly prepared 2,2%-azino-di-(3-ethylbenzthiazoline sulfonate)peroxide solution was added and the optical density was measured.
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Fig. 1. Mouse anti-human IFN-a and -b antisera recognized rsgp41 and ISP in ELISA-assay. Fig. 1a show crossreaction of anti-IFN-a and -b antisera (lane A – H) and show control sera (normal mouse sera) (lane I – L) with rsgp41; Fig. 1b show cross-reaction of anti-IFN-a and -b antisera (lane A – D) and control serum (lane E and F) with ISP. Coating of antigen: rsgp41 (2 mg/ml) (Fig. 1a); ISP (4 mg/ml) (Fig. 1b). AS-alpha-1 and AS-alpha-2: anti-IFN-a antisera from two mice (1:20 and 1:100 dilutions). AS-beta-1 and AS-beta-2: anti-IFN-b antisera from two mice (1:20 and 1:100 dilutions). NMS, normal mouse serum (1:20 and 1:100 dilutions). The figure shows data from three separate experiments.
3. Results and discussion Recently we have reported that a common epitope exists between IFN-a, -b and HIV-1 gp41 [15], and the common epitope was located in the receptor-binding regions of gp41 and of human type I interferon, namely, in the immunosuppressive domain (ISD, aa583–599) of gp41 and in two regions (aa29 – 35 and 123 – 140) of human IFN-a, -b and -v, which form IFN-a/b-receptor binding domains [15]. To clarify the functional mechanism of IFN-a-vaccine and relationship between human type I interferon and HIV-1 gp41, we prepared antibodies against IFN-a, -b and gp41, and examined crossreaction of these antibodies. Mouse antisera to IFN-a and -b could recognize rsgp41 (aa539–684) (Fig. 1a, A – H), while two normal mouse sera (pre-immune sera) did not, and showed only a background (Fig. 1a, I – L). Moreover, the antisera could recognize immunosuppressive peptide (aa583– 599) of gp41 (Fig. 1b, A – D). Besides, mouse antiserum against rsgp41 could bind human IFN-a and -b (Fig. 2). These results indicate that the common immunological epitope exists in human IFN-a, -b and gp41 ISP, suggesting that the antibodies induced in HIV-infected patients by IFN-a-vaccine could bind gp41 ISP. In fact, Gringeri had observed that HIV-infected patients treated with a IFN-a-vaccine showed increase of significant antibody titer against gp41 peptide aa560 – 599 and a significant reduction of disease progression [11], providing an important clinical evidence for our findings. Based on the results that antisera to human IFN-a and -b crossreacted with rsgp41 (Fig. 1a) and ISP (Fig. 1b), we examined whether the antisera to human IFN-a and -b like anti-rsgp41 antiserum inhibit binding of the putative receptor protein P45 to rsgp41 in SPR-assay.
Rsgp41 was coated to slide (Fig. 3, line A–B). Then the sample cell was washed by PBS (Fig. 3, line B–C), and normal mouse serum (NMS) (Fig. 3a, b, c, line C–D) or gelatine (Fig. 3d, line C–D) were added to block nonspecific binding. After washing by PBS (Fig. 3, line D–E), the putative receptor protein p45 was pumped into the sample cell. The line F–G shows the case after washing by PBS. The result indicates clearly that rsgp41 interacts with the putative receptor protein P45 (Fig. 3, line E–F). In the inhibition experiments, after addition of mouse antisera to human IFN-a and b and rsgp41 (instead of normal mouse serum or gelatine), the adsorption (Fig. 3a, line C1 –D1; Fig. 3b, C2 –D2; Fig. 3c, C3 –D3) showed stronger then the adsorption the ease of normal mouse serum (Fig. 3a, b, c, line C–D),and the protein p45 did not adsorb (Fig. 3a, line E1 –F1;
Fig. 2. Mouse anti-gp41 antiserum recognized IFN-a and -b in ELISA-assay. NMS, normal mouse serum; AS, mouse anti-rsgp41 serum. Coating of antigen: IFN-a (2 mg/ml); IFN-b (2 mg/ml). The figure shows data from two separate experiments.
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Fig. 3. Inhibition of gp41-binding to the putative receptor protein p45 by blocking the receptor binding site (aa583 – 599) of gp41 using antisera to human IFN-a (Fig. 3a), -b (Fig. 3b) and rsgp41 (Fig. 3c) in SPR-assay. NMS, normal mouse serum. AMS-IFN-a, mouse antiserum to IFN-a. AMS-IFN-b, mouse antiserum to IFN-b. AMS-gp41, mouse antiserum to rsgp41. The lines noted absorptions of proteins between 0 and 260 min. Lines A – B, A1 – B1, A2 – B2 and A3 –B3, absorption of gp41 to slide. Lines B – C, B1 – C1, B2 – C2 and B3 – C3, adsorption after washing using PBS. C–D, normal mouse serum (pre-immune serum); C1 –D1, adsorption of antiserum to IFN-a; C2 – D2: adsorption of antiserum to IFN-b; C3 –D3, adsorption of antiserum to rsgp41. Lines D–E, D1 –E1, D2 –E2 and D3 – E3, adsorption after washing using PBS. Lines E – F, E1 – F1, E2 –F2 and E3 –F3, adsorption of p45. Lines F–G, F1 –G1, F2 –G2 and F3 – G3, adsorption after washing using PBS. Fig. 3d shows p45-binding to rsgp41 by using gelatine to block nonspecific binding.
Fig. 3b, E2 –F2; Fig. 3c, E3 – F3), suggesting that antisera to human IFN-a, -b and rsgp41 could inhibit binding of the putative receptor protein p45 to gp41. The inhibition of p45-binding to gp41 by the antisera to IFN-a and -b is due to blocking the receptor binding
site (ISD, aa583–599) on gp41, because the antiserums to IFN-a and -b could recognize gp41 ISP (aa583–599) (Fig. 1b). The result of this test suggests that the therapeutic effect of IFN-a-vaccine on HIV-infected patients may be associated with the inhibition of gp41-
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binding to the putative cellular receptor protein p45 by the antibodies recognizing this common immunological epitope in the receptor binding region (immunosuppressive domain, aa583– 599) in gp41. Recent studies have demonstrated a critical step in the HIV entry mechanism that structural rearrangements in the gp41 core structure occur after gp120 binding to its receptors which induce exposure of the gp41 heptad repeat region [19,20], and binding of DP-178 peptide to the gp41 heptad repeat region can block the virus-dependent fusion [19,20], suggesting that the gp41 heptad repeat region (containing the common immunological epitope) is a critical structure for an effective vaccine.
Acknowledgements This work was supported by the 863-program, National Natural Science Foundation of China (Project NSFC-39770696 and NSFC-39880043), and by the China–Austria Cooperation (project V.B. 1).
References [1] G. Alkhatib, C. Combadiere, C.C. Broder, et al., Science 272 (1996) 1955 – 1958.
.
257
[2] P.L.St.J. Jones, T. Korte, et al., J. Biol. Chem. 273 (1998) 404 – 409. [3] W. Weissenhorn, A. Dessen, et al., Nature 387 (1997) 426–430. [4] S. Vanini, R. Longhi, A. Lazzarin, et al., AIDS 7 (1993) 167– 174. [5] S. Jiang, K. Lin, N. Strick, et al., Nature (Lond.) 365 (1993) 113. [6] M.G. Lewis, W.R. Elkins, F.E. McCutchan, et al., Vaccine 11 (1993) 1347 – 1355. [7] Y.H. Chen, C. Ebenbichler, R. Vornhagen, et al., AIDS 6 (1992) 533 – 539. [8] L.A. Henderson, M.N. Qureshi, J. Biol. Chem. 268 (1993) 15291 – 15297. [9] Y.H. Chen, M.P. Dierich, Immunol. Lett. 52 (1996) 153–156. [10] D. Zagury, I. Gagne, B. Reveil, et al., Lancet 2 (1985) 449. [11] A. Gringeri, E. Santagostino, P.M. Mannucci, et al., J. AIDS 7 (1994) 978 – 988. [12] D. Zagury, J. Bernard, A. Halbreich, et al., J. AIDS 5 (1992) 676 – 681. [13] Y.H. Chen, H. Stoiber, M.P. Dierich, Immunol. Lett. 55 (1997) 15 – 18. [14] Y.H. Chen, J.N. Feng, G. Stockl, et al., Molec. Immunol. 34 (1998) 1259 – 1262. [15] Y.H. Chen, M.P. Dierich, Immunol. Today 19 (1998) 586–587. [16] Y.H. Chen, G. Bock, R. Vornhagen, et al., Mol. Immunol. 31 (1994) 977 – 982. [17] M.C. Dubs, D. Altschuh, M.H.V. Van Regenmortel, Immunol. Lett. 31 (1991) 59 – 64. [18] Z. Liu, H. Qin, C. Xiao, et al., Eur. Biophys. J. 24 (1995) 31–38. [19] R.A. Furuta, C.T. Wild, Y. Weng, C.D. Weiss, Nature Stuct. Biol. 5 (1998) 276 – 279. [20] D.C. Chan, P.S. Kim, Cell 93 (1998) 681 – 684.