Kirsten A. Nagashima

University of Amsterdam, Amsterdamo, North Holland, Netherlands

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Publications (12)109.65 Total impact

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    ABSTRACT: The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.
    The Journal of Infectious Diseases 06/2012; 206(5):706-13. DOI:10.1093/infdis/jis416 · 5.78 Impact Factor
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    ABSTRACT: Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4(+) T cells, in a cell-type-dependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.
    Journal of Virology 06/2011; 85(16):8227-40. DOI:10.1128/JVI.00767-11 · 4.65 Impact Factor
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    ABSTRACT: Viral entry inhibitors represent an emerging mode of therapy for human immunodeficiency virus type 1 (HIV-1) infection. PRO 542 (CD4-immunoglobulin G2) is a tetravalent CD4-immunoglobulin fusion protein that broadly neutralizes primary HIV-1 isolates. PRO 542 binds to the viral surface glycoprotein gp120 and blocks attachment and entry of virus into CD4(+) cells. Previously, PRO 542 demonstrated antiviral activity without significant toxicity when tested at single doses ranging to 10 mg/kg. In this study, 12 HIV-infected individuals were treated with 25-mg/kg single-dose PRO 542 and then monitored for safety, antiviral effects, and PRO 542 pharmacokinetics for 6 weeks. The study examined two treatment cohorts that differed in the extent of HIV-1 disease progression. PRO 542 at 25 mg/kg was well tolerated and demonstrated a serum half-life of 3 days. Statistically significant acute reductions in HIV-1 RNA levels were observed across all study patients, and greater antiviral effects were observed in the cohort of patients with more advanced HIV-1 disease. In advanced disease (HIV-1 RNA > 100,000 copies/ml; CD4 lymphocytes < 200 cells/mm(3)), PRO 542 mediated an 80% response rate and statistically significant approximately 0.5 log(10) mean reductions in viral load for 4 to 6 weeks posttreatment. Similar findings were obtained in an analysis of all (n = 11) advanced disease patients treated to date with single doses of PRO 542 ranging from 1 to 25 mg/kg. In addition, a significant correlation was observed between antiviral effects observed in vivo and viral susceptibility to PRO 542 in vitro. The findings support continued development of PRO 542 for salvage therapy of advanced HIV-1 disease.
    Antimicrobial Agents and Chemotherapy 03/2004; 48(2):423-9. DOI:10.1128/AAC.48.2.423-429.2004 · 4.45 Impact Factor
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    ABSTRACT: Background: PRO 542 (CD4-IgG2) is a novel HIV-1 entry inhibitor that comprises the gp120-binding domains of human CD4 fused to the heavy and light chain constant regions of human IgG2. PRO 542 blocks the initial attachment of virus to target cells and has shown favorable tolerability, pharmacology and antiviral effects in Phase I/II clinical testing. Thus far, PRO 542 has been administered intravenously, but subcutaneous (SC) or intramuscular (IM) dosage forms potentially could be self-administered by patients outside a hospital environment. Methods: We performed buffer and excipient screening in order to identify highly concentrated PRO 542 formulations that would be compatible with SC and/or IM dosing. Lead formulations were comparatively evaluated in a battery of biophysical and antiviral assays prior to tolerability and pharmacology testing in animals. Results: Stable liquid formulations containing ≥150 mg/mL PRO 542 were obtained upon optimization of the buffer matrix, resulting in a 30-fold concentration over the existing intravenous (IV) dosage form. In the highly concentrated dosage forms, PRO 542 was fully active in blocking HIV-1 fusion and was free of aggregates or other degradation products. The new dosage forms also demonstrated excellent tolerability and pharmacokinetics when given by IV, IM and SC routes. Conclusions: Highly concentrated dosage forms of PRO 542 demonstrate promising preclinical properties that support advancement into human testing. The new SC and IM dosage forms may enable patients to self-administer antiviral amounts of PRO 542 on an infrequent basis.
    Infectious Diseases Society of America 2003 Annual Meeting; 10/2003
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    ABSTRACT: Background: The chemokine receptor CCR5 is a requisite fusion coreceptor for primary HIV-1 isolates and provides a promising target for a new generation of antiretroviral agents. PRO 140 is a humanized anti-CCR5 monoclonal antibody (mAb) that broadly and potently blocks CCR5-mediated HIV-1 entry without CCR5 antagonism, thus offering a distinct therapeutic profile compared to small-molecule CCR5 antagonists in development. Immunotoxicology is an emerging field that examines the potential impact of drugs on immune system function. Here we report the findings of immunotoxicology studies performed using PRO 140 prior to initiation of Phase 1 clinical testing. Methods: PRO 140 was tested in a battery of in vitro assays of immune system function mediated by chemokine and non-chemokine mechanisms, and the immunologic activity of PRO 140 was compared with its antiviral activity. Results: At concentrations that provide complete control of HIV-1 replication (~4 μg/mL), PRO 140 had no effect on CCR5 signaling in response to CC-chemokines. Similarly, at concentrations ranging to 100 μg/mL, PRO 140 had no effect on lymphocyte proliferation in response to mitogenic and allogeneic stimulation. Consistent with its IgG4,κ subtype, PRO 140 did not demonstrate significant binding to cells that express high levels of FcγR1 (CD64) and other Fc receptors. Lastly, PRO 140 did not mediate significant levels of antibody-dependent cellular cytotoxicity or complement-dependent lysis of CCR5-expressing target cells. Conclusions: PRO 140 did not interfere with normal immune system function in vitro, consistent with its lack of CCR5 antagonism and Fc-mediated effector activity. As an immunologically silent inhibitor of CCR5-mediated HIV-1 entry, PRO 140 may offer distinct tolerability and therapeutic profiles in man.
    Infectious Diseases Society of America 2003 Annual Meeting; 10/2003
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) entry proceeds via a cascade of events that afford promising targets for therapy. PRO 542 neutralizes HIV-1 by blocking its attachment to CD4 cells, and T-20 blocks gp41-mediated fusion. Both drugs have shown promise in phase 1/2 clinical trials. Here, the drugs were tested individually and in combination in preclinical models of HIV-1 infection, and inhibition data were analyzed for cooperativity by using the combination index method. Synergistic inhibition of virus-cell and cell-cell fusion was observed for phenotypically diverse viruses for a broad range of drug concentrations, often resulting in > or = 10-fold dose reductions in vitro. Additional mechanism-of-action studies probed the molecular basis of the synergies. The markedly enhanced activity observed for the PRO 542:T-20 combination indicates that the multistep nature of HIV-1 entry leaves the virus particularly vulnerable to combinations of entry inhibitors. These findings provide a strong rationale for evaluating combinations of these promising agents for therapy in vivo.
    The Journal of Infectious Diseases 05/2001; 183(7):1121-5. DOI:10.1086/319284 · 5.78 Impact Factor
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    ABSTRACT: CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.
    Journal of Virology 02/2001; 75(2):579-88. DOI:10.1128/JVI.75.2.579-588.2001 · 4.65 Impact Factor
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    ABSTRACT: The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.
    Journal of Virology 06/1999; 73(5):4145-55. · 4.65 Impact Factor
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    ABSTRACT: The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.
    Journal of Virology 02/1998; 72(1):279-85. · 4.65 Impact Factor
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    ABSTRACT: The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.
    Nature Medicine 02/1998; 4(1):72-7. DOI:10.1038/nm0198-072 · 28.05 Impact Factor
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    ABSTRACT: Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.
    Journal of Virology 10/1996; 70(9):6437-41. · 4.65 Impact Factor
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    ABSTRACT: The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.
    Nature 07/1996; 381(6584):667-73. DOI:10.1038/381667a0 · 42.35 Impact Factor

Publication Stats

4k Citations
109.65 Total Impact Points

Institutions

  • 2011
    • University of Amsterdam
      • Department of Medical Microbiology
      Amsterdamo, North Holland, Netherlands
  • 2004
    • Beth Israel Medical Center
      New York City, New York, United States
  • 2003
    • Progenics Pharmaceuticals, Inc.
      Tarrytown, New York, United States
  • 2001
    • Africa Institute of South Africa
      Πρετόρια/Πόλη του Ακρωτηρίου, Gauteng, South Africa
  • 1999
    • The Rockefeller University
      • Aaron Diamond AIDS Research Center (ADARC)
      New York, New York, United States