In Vitro Evaluation of Experimental Agents for Anti‐HIV Activity

Douglas D. Richman1, Victoria A. Johnson2, Douglas M. Mayers3, Takuma Shirasaka4, Mary C. O'Brien4, Hiroaki Mitsuya4

1 University of California, San Diego, La Jolla, California, 2 University of Alabama at Birmingham, Birmingham, Alabama, 3 Walter Reed Army Institute of Research, Rockville, Maryland, 4 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.9
DOI:  10.1002/0471142735.im1209s08
Online Posting Date:  May, 2001
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Abstract

This unit presents an assay that has proven useful as an initial screening test is an HIV cytopathic effect (CPE) inhibition assay in which immortalized T cell lines (e.g., ATH8 or MT2) that are profoundly sensitive to the cytopathic effect of certain strains of HIV are utilized as target cells. Additional protocols assess the antiā€HIV activity of certain candidate agents by measuring inhibition of syncytium formation or p24 gag protein production by ELISA. Calculation of the 50% inhibitory concentration (IC50) is also presented.

     
 
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Table of Contents

  • Basic Protocol 1: Protection of Target T Cells Against the Cytopathic Effect of HIV
  • Basic Protocol 2: Inhibition of Syncytial Focus (Plaque) Formation in CD4‐Expressing HeLa Cells
  • Basic Protocol 3: Inhibition of HIV p24 Antigen as Assessed by Production in Peripheral Blood Mononuclear Cells by ELISA
  • Support Protocol 1: Calculation of 50% Inhibitory Concentration (IC50)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protection of Target T Cells Against the Cytopathic Effect of HIV

  Materials
  • ATH8 (HTLV‐1 transformed tetanus‐toxoid‐specific CD4+ T cell clone; Mitsuya et al., ) or similar target T cells susceptible to the cytopathic effect of HIV
  • IL‐2‐supplemented culture medium appropriate for cell line (see )
  • Cell‐free HIV preparation or γ‐irradiated permissively HIV‐1‐infected cells (AIDS Research and Reagent Reference Program), prepared as described in units 12.2 12.4
  • Antiviral agent to be tested
  • 50‐ml polypropylene conical tubes (Falcon #2070)
  • 10‐ml polystyrene tissue culture tubes (Falcon #3033)
  • Sorvall #RC3B centrifuge equipped with H‐2000 rotor, or equivalent
  • Hemacytometer
  • Additional reagents and equipment for quantitation of HIV (unit 12.2) and counting cells using a hemacytometer and determining cell viability by trypan blue exclusion ( appendix 33)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Inhibition of Syncytial Focus (Plaque) Formation in CD4‐Expressing HeLa Cells

  Materials
  • HT4‐6C cells (AIDS Research and Reference Reagent Program)
  • Trypsin‐EDTA (GIBCO/BRL)
  • Maintenance medium: complete DMEM‐10 ( appendix 2A)
  • Plaque assay medium, containing HIV stock titered by serial dilution (unit 12.2) in HT4‐6C cells or antiviral agent to be tested
  • 100% MeOH
  • 0.3% (v/v) crystal violet in distilled water, filtered (e.g., using a coffee filter)
  • 70% ethanol
  • 24‐well tissue culture plates (Falcon #3047)
  • Inverted microscope
  • Additional reagents and equipment for evaluation of syncytia formation (unit 12.2)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Inhibition of HIV p24 Antigen as Assessed by Production in Peripheral Blood Mononuclear Cells by ELISA

  Materials
  • Phytohemagglutinin (PHA)‐stimulated PBMC from HIV‐seronegative (normal) donors
  • IL‐2‐supplemented culture medium containing 5% (v/v) IL‐2
  • recipePhosphate‐buffered saline (PBS; appendix 2A), sterile
  • Virus‐containing supernatant, generated from co‐culture of PBMC from HIV‐seropositive blood with PHA‐stimulated PBMC from HIV‐seronegative (normal) donors (unit 12.2)
  • Disinfectant (e.g., 10% bleach, unit 12.1)
  • Wash medium: RPMI 1640 medium (GIBCO/BRL), without serum or other additives
  • 1 mM AZT stock solution, prepared in sterile PBS and stored at−20°C
  • 96‐well flat‐bottom or round‐bottom microtiter plates
  • p24 ELISA plate (unit 12.5)
  • Additional reagents and equipment for evaluating cytopathic effects (unit 12.2), determining cell viability by trypan blue exclusion ( appendix 33) and detecting HIV proteins by ELISA (unit 12.5)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

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Key References
   Johnson, V.A. 1992. New Developments in Antiretroviral Drug Therapy for HIV Infection. In AIDS Clinical Review, (Volberding, P., Jacobson, M.A. eds.) pp. 69‐104. Marcel Dekker, New York.
  Reviews of this rapidly changing field—the first three are as of late 1991, and the last is as of late 1992.
   Johnston, M.I. and Hoth, F.D. 1993. Present status and future prospects for HIV therapies. Science (Wash. DC) 260:1286‐1293.
   Mitsuya, H., Yarchoan, R., Kageyama, S., and Broder, S. 1991. Targeted therapy of human immunodeficiency virus‐related disease. FASEB J. 5:2369‐2381.
   Richman, D. 1993. Resistance of clinical isolates of human immunodeficiency virus to antiretroviral agents. Antimicrob. Agents Chemother. 37:1207:1213.
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