A Novel Cyclic Peptide Immunization Strategy for Preventing HIV-1/AIDS Infection and Progression
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m301209200
ISSN1083-351X
AutoresShogo Misumi, Masafumi Endo, Ryouzaburou Mukai, Kuniomi Tachibana, Mamoru Umeda, Tetsuro Honda, Nobutoki Takamune, Shozo Shoji,
Tópico(s)vaccines and immunoinformatics approaches
ResumoA novel synthetic peptide immunogen targeting the human immunodeficiency virus type-1 (HIV-1) coreceptor CXCR4 was evaluated for its capacity to induce CXCR4-specific antibodies with anti-HIV-1 activity in BALB/c mice and cynomolgus monkeys. A cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4 (cDDX4) was prepared in which Gly-Asp, as the dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (linear DDX4, Asn176 to Ile185) derived from the undecapeptidyl arch (UPA; Asn176 to Cys186) of extracellular loop 2 (ECL-2) in CXCR4. Immunization of BALB/c mice with cDDX4 conjugated with a multiple-antigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies, and monoclonal antibody IA2-F9 reacted with cDDX4, but not with linear DDX4, as determined by real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CXCR4 but not with cells expressing the other HIV coreceptor, CCR5. Furthermore, the antibody inhibited the replication of HIV-1 X4 virus (using CXCR4), as shown by an infection assay using both MAGIC-5 cells and MT4 cells, but not that of HIV-1 R5 virus (using CCR5). The antibody weakly interfered with chemotaxis induced by stromal cell-derived factor-1α in THP-1 cells or moderately inhibited the chemotaxis of Molt4#8 cells under the same conditions. In addition, immunization of cynomolgus monkeys also induced cDDX4-specific antibodies with anti-HIV activity. Taken together, these results indicate that cDDX4 conjugated with a multi-antigen peptide induces the conformational epitope-specific antibodies to the undecapeptidyl arch of CXCR4 may be a novel candidate immunogen for preventing disease progression in HIV-1-infected individuals. A novel synthetic peptide immunogen targeting the human immunodeficiency virus type-1 (HIV-1) coreceptor CXCR4 was evaluated for its capacity to induce CXCR4-specific antibodies with anti-HIV-1 activity in BALB/c mice and cynomolgus monkeys. A cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4 (cDDX4) was prepared in which Gly-Asp, as the dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (linear DDX4, Asn176 to Ile185) derived from the undecapeptidyl arch (UPA; Asn176 to Cys186) of extracellular loop 2 (ECL-2) in CXCR4. Immunization of BALB/c mice with cDDX4 conjugated with a multiple-antigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies, and monoclonal antibody IA2-F9 reacted with cDDX4, but not with linear DDX4, as determined by real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CXCR4 but not with cells expressing the other HIV coreceptor, CCR5. Furthermore, the antibody inhibited the replication of HIV-1 X4 virus (using CXCR4), as shown by an infection assay using both MAGIC-5 cells and MT4 cells, but not that of HIV-1 R5 virus (using CCR5). The antibody weakly interfered with chemotaxis induced by stromal cell-derived factor-1α in THP-1 cells or moderately inhibited the chemotaxis of Molt4#8 cells under the same conditions. In addition, immunization of cynomolgus monkeys also induced cDDX4-specific antibodies with anti-HIV activity. Taken together, these results indicate that cDDX4 conjugated with a multi-antigen peptide induces the conformational epitope-specific antibodies to the undecapeptidyl arch of CXCR4 may be a novel candidate immunogen for preventing disease progression in HIV-1-infected individuals. Human immunodeficiency virus type-1 (HIV-1) 1The abbreviations used are: HIV, human immunodeficiency virus; cDDX4, cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4; cDDX4-MAP, cDDX4 conjugated with a multiple-antigen peptide; ECL, extracellular loop; HAART, highly active antiretroviral therapy; UPA, undecapeptidyl arch; SDF-1α, stromal cell-derived factor-1α; PBMCs, peripheral blood mononuclear cells; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; MOE, molecular operating environment; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; MAP, multiple-antigen peptide; FCS, fetal calf serum; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection.1The abbreviations used are: HIV, human immunodeficiency virus; cDDX4, cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4; cDDX4-MAP, cDDX4 conjugated with a multiple-antigen peptide; ECL, extracellular loop; HAART, highly active antiretroviral therapy; UPA, undecapeptidyl arch; SDF-1α, stromal cell-derived factor-1α; PBMCs, peripheral blood mononuclear cells; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; MOE, molecular operating environment; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; MAP, multiple-antigen peptide; FCS, fetal calf serum; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection. requires both CD4 and a chemokine receptor for cellular entry. Following its binding to CD4, the viral envelope protein changes its conformation to bind to the chemokine receptor and initiates fusion with the cellular membrane (1Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1865) Google Scholar, 2Bieniasz P.D. Cullen B.R. Front. Biosci. 1998; 3: 44-58Crossref PubMed Google Scholar, 3Moore J.P. Trkola A. Dragic T. Curr. Opin. Immunol. 1997; 9: 551-562Crossref PubMed Scopus (448) Google Scholar, 4Littman D.R. Cell. 1998; 93: 677-680Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Because chemokine receptors CCR5 and CXCR4 are the main coreceptors for cellular entry of HIV-1, viral strains are classified as R5, X4, or R5X4 according to the usage of chemokine receptor (5Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3614) Google Scholar, 6Berger E.A. Doms R.W. Fenyo E.M. Korber B.T. Littman D.R. Moore J.P. Sattentau Q.J. 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Therefore, development of an antiviral drug that targets novel molecules is desirable (13Takamune N. Hamada H. Misumi S. Shoji S. FEBS Lett. 2002; 527: 138Crossref PubMed Scopus (20) Google Scholar). CXCR4 antagonists that prevent entry of X4 virus are one of the candidates. Some studies showed that blockade of CXCR4 can possibly prevent emergence of X4 virus and change the phenotype of already existing X4 virus to R5 virus (14Este J.A. Cabrera C. Blanco J. Gutierrez A. Bridger G. Henson G. Clotet B Schols D. De Clercq E. J. Virol. 1999; 73: 5577-5585Crossref PubMed Google Scholar, 15Philpott S. Weiser B. Anastos K. Kitchen C.M. Robison 3rd, E. Meyer W.A. Sacks H.S. Mathur-Wagh U. Brunner C. Burger H. J. Clin. Invest. 2001; 107: 431-438Crossref PubMed Scopus (77) Google Scholar). In addition, a recent statistical study showed that long term nonprogressors have high levels of plasma stromal cell-derived factor-1α (SDF-1α), which is a ligand for CXCR4, and low CXCR4 expression levels on T lymphocytes; in advancing disease, the expression level of CXCR4 increases (16Soriano A. Martinez C. Garcia F. Plana M. Palou E. Lejeune M. Arostegui J.I. DeLazzari E. Rodriguez C. Barrasa A. Lorenzo J.I. Alcami J. DelRomero J. Miro J.M. Gallart T. J. Infect. Dis. 2002; 186: 922-931Crossref PubMed Scopus (110) Google Scholar). These reports indicate that CXCR4 is an attractive target, which not only inhibits the entry of X4 virus but may also delay the disease progression to AIDS, and may become the target of immunotherapeutic approaches. CXCR4 has been shown to be critical for development (17Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9448-9453Crossref PubMed Scopus (1402) Google Scholar, 18Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y Kitamura Y. Matsushima K. Yoshida N. Nishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-594Crossref PubMed Scopus (1310) Google Scholar, 19Zou Y.R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2100) Google Scholar) but is probably dispensable in adults. However, when developing a self-antigen as a target of an immunotherapeutic approach, unexpected effects must be taken into consideration together with sufficient immune responses. Hence, it is desirable to design an immunogen that targets a restricted protein region rather than the whole protein. Therefore, a linear peptide immunogen has the advantage in that it can elicit a desired immune response against a restricted protein region, whereas it can hardly induce antibodies against the conformational epitope (20Arnon R. Horwitz R.J. Curr. 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Therefore, the antibody induced by a linear synthetic peptide against the extracellular loop region of CXCR4 could recognize a denatured protein but could not recognize the native protein. A linear peptide is inadequate for mimicking the conformational loop region of CXCR4, and mimicking the native conformational epitope by a peptide is critical for inducing the antibody against a conformational epitope. Therefore, to use the peptide immunogen for an immunotherapeutic approach targeting CXCR4, it is essential for the designed peptide immunogen to target not only the restricted region of CXCR4 but also to mimic the conformational epitope of the targeted region. In this study, we investigated the application of a cyclic dodecapeptide peptide (cDDX4) that was designed to mimic the native conformational epitope of the undecapeptidyl arch (UPA) (24Misumi S. Nakajima R. Takamune N. Shoji S. J. Virol. 2001; 75: 11614-11620Crossref PubMed Scopus (22) Google Scholar, 25Nardin E.H. Oliveira G.A. Calvo-Calle J.M. Castro Z.R. Nussenzweig R.S. Schmeckpeper B. Hall B.F. Diggs C. Bodison S. Edelman R. J. Infect. Dis. 2000; 5: 1486-1496Crossref Scopus (132) Google Scholar) in CXCR4 for use as a novel immunotherapy for AIDS. Immunization with cDDX4 conjugated with a multiantigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies that preferentially recognized the cyclic structure of the antigen and cross-reacted with cell surface CXCR4. In addition, the induced antibody inhibited the replication of HIV-1 X4 virus but not that of HIV-1 R5 virus as determined by infection assay using MAGIC-5 cells and by a productive infection assay using MT4 cells. These results indicate that cDDX4 sufficiently mimics the conformational epitope of UPA in cell surface CXCR4 and possibly induces antibodies that inhibit cellular entry of HIV-1. In addition, immunization of cynomolgus monkeys with cDDX4-MAP also elicited cDDX4-specific antibodies with anti-HIV activity. Taken together, we propose that an immunotherapeutic approach using cDDX4-MAP as the self-antigen immunogen may be a novel strategy for AIDS therapy. Cell Culture—The human T-lymphotropic virus type I-infected cell line (MT4), human T cell lines (CEM and Molt4#8), human monocytic cell lines (THP-1 and U937), and peripheral blood mononuclear cells (PBMCs) were cultured at 37 °C in 5% (v/v) CO2 in the RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). MAGIC-5 cells and CD4-transduced human glioma cell lines (NP2/CD4, NP2/CD4/CXCR4, and NP2/CD4/CCR5) were cultured at 37 °C in 5% (v/v) CO2 in Dulbecco's modified Eagle's medium supplemented with 2.5% or 10% FCS, respectively. Anti-CXCR4 Antibody Generation—The antigen and antibody were prepared using the protocol of Misumi et al. (24Misumi S. Nakajima R. Takamune N. Shoji S. J. Virol. 2001; 75: 11614-11620Crossref PubMed Scopus (22) Google Scholar). To mimic the native conformational epitope of human CXCR4, the CXCR4-derived linear dodecapeptide (linear DDX4: N2H-DNVSEADDRYIG-COOH) was synthesized using an automatic peptide synthesizer and then cyclized. The cyclic dodecapeptide (cDDX4: DNVSEADDRYIG) was conjugated with a multiple-antigen peptide (MAP) through the formation of the peptide bound between the β-carboxyl group of Asp within the cDDX4 and the amino group within MAP. MAP, which is composed of 2-fold bifurcating polylysine core developed as a carrier of a peptide antigen, is capable of eliciting a strong antibody response in mice, monkeys, and humans (25Nardin E.H. Oliveira G.A. Calvo-Calle J.M. Castro Z.R. Nussenzweig R.S. Schmeckpeper B. Hall B.F. Diggs C. Bodison S. Edelman R. J. Infect. Dis. 2000; 5: 1486-1496Crossref Scopus (132) Google Scholar). All peptides were purified by high-performance liquid chromatography (Waters), and their molecular masses were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Burker Franzen Analytik GmbH, Bremen, Germany). Female BALB/c mice were immunized with the cDDX4-MAP conjugate using the protocol of Galfre and Milstein (26Galfre G. Milstein C. Methods Enzymol. 1981; 73: 3-46Crossref PubMed Scopus (1392) Google Scholar). The resulting hybridoma that produced the most potent supernatant was screened in 96-well plates for the reactivity to Multi-Pin Block (Chiron Technologies), which was conjugated with cDDX4 and was cloned by limiting dilution. The anti-CXCR4 antibody, IA2-F9, was recovered by ammonium sulfate precipitation and purified using a Sephadex G-150 column (Amersham Biosciences). Real-time Biomolecular Interaction Analysis using Surface Plasmon Resonance—Recognition of the cyclic structure of cDDX4 by IA2-F9 was analyzed by the surface plasmon response technology using BIA-core2000 as described previously (24Misumi S. Nakajima R. Takamune N. Shoji S. J. Virol. 2001; 75: 11614-11620Crossref PubMed Scopus (22) Google Scholar, 27Bernard A. Bosshard H.R. Eur. J. Biochem. 1995; 230: 416-423Crossref PubMed Scopus (58) Google Scholar, 28MacKenzie C.R. Hirama T. Deng S.J. Bundle D.R. Narang S.A. Young N.M. J. Biol. Chem. 1996; 271: 1527-1533Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The free β-carboxyl group of Asp that was used as a spacer-armed dipeptide in cDDX4 was conjugated to 5-[5-(N-succinimidyloxycarbonyl)pentylamido]hexyl d-biotinamide through ethylenediamine. Biotinylated cDDX4 was immobilized to streptavidin-coated sensor chips. For competition assay, purified IA2-F9 was pretreated with cDDX4 (1 and 10 nmol) or linear DDX4 (1 and 10 nmol) at room temperature prior to BIAcore analysis. All the antigen-antibody interactions were analyzed in a binding buffer (0.02% KH2PO4, 0.29% Na2HPO4, 12H2O, 0.8% NaCl, and 0.02% KCl) at a constant flow rate of 50 μl/min and a constant temperature of 25 °C. The bound antibody was eluted from the biotinylated cDDX4 by a short pulse (20 μl) of 10 mm Gly-HCl (pH 2.0). This regeneration procedure did not alter the ability of cDDX4 to bind to the antibody in subsequent cycles. Kinetic analysis was performed on BIAcore2000 using BIAcore evaluation software. For detecting anti-cDDX4 antibodies in sera derived from immunized cynomolgus monkeys, the sera were dialyzed using Spectra/Por (cutoff molecular masses, 100,000; Spectrum Laboratories Inc.) according to the manufacturer's instructions and analyzed. Antibody and Flow Cytometry—The following antibodies were used: an anti-CXCR4 mAb (clone 12G5, BD Biosciences), an isotype-matched control antibody (Sigma), purified IA2-F9, and fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G or anti-mouse IgM. Cells (1 × 105) were washed with washing buffer (PBS containing 2% FCS and 0.02% NaN3) and incubated with 10 μg/ml primary antibody (purified IA2-F9 or 12G5) for 30 min at 4 °C. The cells were then washed with the same washing buffer and incubated with FITC-conjugated anti-mouse IgG or anti-mouse IgM. Then the cells were washed again and analyzed using an EPICS XL flow cytometer (Beckman Coulter). Chemotaxis Assay—The migration of Molt4#8 and THP-1 was assayed in 24-well cell-culture chambers using an insert with 5.0-μm pore membranes (Corning, Corning, NY) according to the protocol of Gosling et al. (29Gosling J. Monteclaro F.S. Atchison R.E. Arai H. Tsou C.L. Goldsmith M.A. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5061-5066Crossref PubMed Scopus (160) Google Scholar) with slight modifications. Cells (5 × 106) were pretreated with or without IA2-F9 for 30 min and were placed in the upper chamber. Chemotaxis was conducted in the presence of 10 nm SDF-1α (placed in the lower chamber). After incubation for 3 h at 37 °C, cells that migrated from the upper chamber to the lower chamber were quantified by trypan blue dye exclusion. MAGIC-5 Assay—The antiviral activity of IA2-F9 was determined by an infection assay using MAGIC-5 cells (32Hachiya A. Aizawa-Matsuoka S. Tanaka M. Takahashi Y. Ida S. Gatanaga H. Hirabayashi Y. Kojima A. Tatsumi M. Oka S. Antimicrob. Agents Chemother. 2001; 45: 495-501Crossref PubMed Scopus (80) Google Scholar). Because MAGIC-5 cells express CD4, CCR5, and CXCR4, they are susceptible to infection by R5, X4, and R5X4 viruses. The cells were seeded (1 × 104 cells per well) and cultured in a 48-well plate for 24 h. After removal of the medium from each well, the cells were incubated for 30 min with IA2-F9 at the indicated concentrations and were infected with the virus in the presence of DEAE-dextran (20 μg/ml) for 2 h and washed with the culture medium. The cells were cultured in the medium containing IA2-F9 for 48 h, fixed with 1% formaldehyde-0.2% glutaraldehyde in PBS for 5 min, washed, and then stained with X-gal. The number of blue-stained cells was counted under a light microscope. Control experiments were carried out under identical conditions in the absence of the antibody. For determining anti-HIV activity of sera at 0 and 10 weeks derived from immunized cynomolgus monkeys, antibodies were purified from the serum using an NAb™ Protein L Spin kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. For determining anti-HIV activity of sera at 25 and 27 weeks, sera were dialyzed with PBS(–) using Spectra/Por (cutoff molecular mass, 100 kDa; Spectrum Laboratories Inc.) and adjusted to a concentration corresponding to a 1/10 dilution of sera with PBS(–). Productive Infection Assay—MT4 cells (1 × 105) were preincubated with IA2-F9 at the indicated concentrations for 30 min, and then HIV-1 X4 virus (m.o.i. = 0.01) was inoculated to these cells. After washing three times with PBS, the cells were incubated with IA2-F9 (0, 1, and 10 μg/ml) for 72, 96, and 120 h. Then the culture supernatant of each cell was collected, and the p24 antigen level was measured by antigen-capture enzyme-linked assay using a RETRO-TEK HIV-1 p24 antigen enzyme-linked immunosorbent assay kit (ZeptoMetrix Corp.) according to the manufacturer's instructions. Immunization Schedule—All the cynomolgus monkeys were housed in individual cages and maintained according to the rules and guidelines of the National Institute for Infectious Diseases for experimental animal welfare. Three cynomolgus monkeys, 4–6 years old, were immunized intraperitoneally at 0 and 1 week with 300 μg of cDDX4-MAP in complete Freund's adjuvant and boosted subcutaneously at 6 weeks with 300 μg of cDDX4-MAP in incomplete Freund's adjuvant. Furthermore, these monkeys were reboosted subcutaneously at 25 weeks with 300 μg of cDDX4-MAP in Freund's incomplete adjuvant. Another three cynomolgus monkeys were immunized with MAP following the same immunization schedule. Blood samples were obtained at 0, 10, 25, and 27 weeks, which were then subjected to BIAcore analysis and MAGIC-5 assay. Design and Synthesis of cDDX4 —The hypothetical structure model of CXCR4 was based on its homology with rhodopsin (30Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4971) Google Scholar), and energy-minimized with the Molecular Operating Environment, MOE (Chemical Computing Group Inc., Montreal, Quebec, Canada) (Fig. 1A). The extracellular loop-2 (ECL-2) region of CXCR4, and its structure deduced by MOE (Figs. 1B-1 and 2) has a unique arch structure consisting of 11 amino acid residues (UPA) on the basis of the Cys186 residue bound to a Cys109 residue of ECL-1 by a disulfide bond (Swiss-Prot P30991, Fig. 1B-1). The cDDX4 moiety designed to mimic the native conformational epitope of UPA was generated by cyclization of a decapeptide (176NVSEADDRYI185) derived from the UPA sequence by insertion of a spacer-armed dipeptide (Gly-Asp) (shown in red in Fig. 1C). The deduced structure of cDDX4 (shown in blue) was adopted to the structural model of UPA in CXCR4 using the MOE-Align tool (Chemical Computing Group Inc., Montreal, Quebec, Canada, shown in yellow in Fig. 1D).Fig. 2MALDI-TOF MS spectra of linear DDX4 and cDDX4. The spectra exhibit two peaks at m/z 1317.7 and 1335.2: the upper peak corresponds to the ion derived from linear DDX4, and the lower peak to the ion derived from cDDX4. The matrix was a saturated solution of α-cyano-4-hydroxycinnamic acid in solution of acetonitrile-water (1:2, v/v) containing 0.1% trifluoroacetic acid. The fraction with a molecular mass of 17.5 corresponding to H2O was deleted after cyclizing the linear DDX4 with the peptide bond.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cyclization of the linear peptide was confirmed by molecular mass analysis using MALDI-TOF-MS (Fig. 2). The spectra of linear DDX4 and cDDX4 exhibited major peaks at m/z 1335.2 and 1317.7, respectively. The difference in molecular mass between linear DDX4 and cDDX4 indicates the formation of a peptide bond. The cDDX4 was combined with MAP to enhance immunogenicity and then immunized to BALB/c mice. Conformational Specificity of IA2-F9 —Immunization of BALB/c mice with cDDX4-MAP induced production of high titers of antibodies. Using cDDX4-Multi-Pin Block for a screening antigen, monoclonal antibody IA2-F9 that has the highest enzyme-linked immunosorbent assay titer was established. To analyze the conformational epitope-specific recognition of IA2-F9, the surface plasmon resonance technology using BIA-core2000 was applied. cDDX4 was immobilized to a sensor chip by streptavidin-biotin interaction. IA2-F9 recognized cDDX4 on the sensor chip in a dose-dependent manner (K d = 1.55 × 10–8m) (Fig. 3A). IA2-F9 binding competition was observed in the case of pretreatment of IA2-F9 with cDDX4 (Fig. 3B). In contrast, pretreatment of the linear peptide did not affect the binding of IA2-F9 (Fig. 3C). These results indicate that IA2-F9 is a conformational epitope-specific mAb that can recognize the cyclic structure of the cDDX4 antigen. Binding of IA2-F9 to Cell Surface CXCR4 —Mimicry of a conformational epitope by a peptide is critical for induction of antibodies that can recognize the native protein by a peptide antigen. Paradoxically, only a peptide antigen that mimics the conformational epitope could induce antibodies that react with the native protein. The binding of IA2-F9 to cells expressing CXCR4 was determined by flow cytometry. IA2-F9 could recognize CXCR4 but not CCR5 expressed on NP2/CD4 cells (Fig. 4, A–C). These results indicate that IA2-F9 is a conformational epitope-specific mAb and specifically recognizes cell surface CXCR4. Furthermore, analysis of a panel of cells demonstrated that all cells known to express CXCR4, including MT4, THP-1, MAGIC-5, Molt4#8, U937, and PBMCs, were stained by IA2-F9 (Fig. 4, D–I). Chemotaxis Assay—The effect of IA2-F9 binding on the chemokine receptor activity was investigated by chemotaxis assay using SDF-1α as the chemoattractant. Both human T cell lines Molt4#8 and THP-1 were used with or without IA2-F9. Although each cell line showed different chemotaxic reactivities against SDF-1α, IA2-F9 inhibited weakly (THP-1) or moderately (Molt4#8) the SDF-1α-induced chemotaxis (Fig. 5). Anti-HIV Activity of IA2-F9 —Because ECL-2 is thought to be critical for HIV-1 X4 viral entry (31Zhou N. Luo Z. Luo J. Liu D. Hall J.W. Pomerantz R.J. Huang Z. J. Biol. Chem. 2001; 276: 42826-42833Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), anti-UPA antibodies are expected to inhibit the viral infection. The anti-HIV activity of IA2-F9 was determined by an infection assay using MAG-IC-5 cells (32Hachiya A. Aizawa-Matsuoka S. Tanaka M. Takahashi Y. Ida S. Gatanaga H. Hirabayashi Y. Kojima A. Tatsumi M. Oka S. Antimicrob. Agents Chemother. 2001; 45: 495-501Crossref PubMed Scopus (80) Google Scholar). MAGIC-5 cells were generated from CD4- and CXCR4-positive HeLa-CD4-LTR-β-gal (MAGI) cells by transfection with a CCR5 expression plasmid to confer susceptibility to infection of not only X4 virus but also R5 virus. MAGIC-5 cells were inoculated with LAV-1 (X4 virus), JRFL (R5 virus), or 89.6 (R5X4 virus), in the absence or presence of IA2-F9 at indicated concentrations. IA2-F9 inhibited infection of LAV-1 but not those of JRFL and 89.6 (Fig. 6). To verify the antiviral activity of IA2-F9 in an alternative experiment, the inhibitory effect of IA2-F9 on HIV-1 replication in T cells was investigated. MT4 cells, a human T cell line, were infected with HIV-1 X4 virus, LAV-1 (m.o.i. = 0.01) in the presence of IA2-F9 at varying doses, and the spread of infection was monitored based on the accumulation of p24 antigen in culture supernatants. IA2-F9 at 10 μg/ml inhibited HIV-1 LAV-1 infection at the peak of virus production in the control experiment as shown in Fig. 7. These results indicate that cDDX4-MAP could induce the antibody with anti-HIV activity, and suggest the potential of immunization with cDDX4-MAP in AIDS therapy. Immunogenicity of cDDX4-MAP in Cynomolgus Monkeys—To verify whether cDDX4-MAP could induce CXCR4-specific antibodies with anti-HIV-1 activity in nonhuman primates as well as rodents, an experiment was performed using cynomolgus monkeys immunized following the time schedule shown in Fig. 8A. Three cynomolgus monkeys were immunized with cDDX4-MAP in Freund's complete adjuvant or Freund's incomplete adjuvant by intraperitoneal or subcutaneous injection. Another three cynomolgus monkeys were immunized with MAP as the control. The cDDX4-immobilized BIAcore sensor chip was used for detecting cDDX4-specific antibodies in immunized monkeys. cDDX4-bound antibodies were detected in the sera from three monkeys 10 weeks after the initial immunization (Fig. 8B). On the other hand, no significant responses were detected in MAP-immunized monkeys, which are the control (monkeys 4–6). These results indicate that cDDX4-MAP could induce CXCR4-specific antibodies not only in BALB/c mice but also in cynomolgus monkeys. Anti-HIV Activity of Partially
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