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

  • 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

  • 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

  • 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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Literature Cited

Literature Cited
   Balzarini, J., Pauwels, R., Baba, M., Herdewijn, P., DeClercq, E., Broder, S., and Johns, D. 1988. The in vitro and in vivo anti‐retrovirus activity, and intracellular metabolism of 3′‐azido‐2′,3′‐dideoxythymidine and 2′,3′‐dideoxycytidine are highly dependent on the cell species. Biochem. Pharmacol. 37:897‐903.
   Capon, D.J., Chamow, S.M., Mordenti, J., Martsters, S.A., Gregory, T., Mitsuya, H., Byrn, R.A., Lucas, C., Wurm, F.M., Groopman, J.E., Broder, S., and Smith, D.H. 1989. Designing CD4 immunoadhesins for AIDS therapy. Nature (Lond.) 337:525‐531.
   Chou, T.C. 1991. Synergism and Antagonism in Chemotherapy (T.C. Chou and D.C. Rideout, eds.). Academic Press, San Diego.
   Chou, T.C. and Talalay, P. 1984. Quantitative analysis of dose‐effect relationships: The combined effect of multiple drugs of enzyme inhibitors. Adv. Enzyme Regul. 22:27‐55.
   Daar, E.S., Li, E.L., Moudgil, T., and Ho, D.D. 1990. High concentrations of recombinant soluble CD4 are required to neutralize primary HIV‐1 isolates. Proc. Natl. Acad. Sci. U.S.A. 87:6574‐6579.
   Dahlberg, J.E., Mitsuya, H., Broder, S., and Aaronson, S.A. 1987. 2′, 3′‐dideoxynucleosides: Broad spectrum antiretroviral activity. Proc. Natl. Acad. Sci. U.S.A. 84:2469‐2473.
   DeClercq, E. (ed.) 1990. In “Design of Anti‐AIDS Drugs.” Elsevier/North‐Holland Amsterdam.
   DeClercq, E. 1992. HIV inhibitors targeted at the reverse transcriptase. AIDS Res. Hum. Retroviruses 8:119‐134.
   Gartner, S., Markovits, P., Markovits, D.M., Kaplan, M.H., Gallo, R.C., and Popovic, M. 1988. The role of mononuclear phagocytes in HTLV‐III/LAV infection. Science (Wash. DC), 233:215‐219.
   Gingeras, T.R., Prodanovich, P., Latimer, T., Guatelli, J.C., Richman, D.D., and Barringer, K.J. 1991. Use of self‐sustained sequence replication amplification reaction to analyze and detect mutations in zidovudine‐resistant human immunodeficiency virus. J. Infect. Dis. 164:1066‐1074.
   Haertle, T., Carrera, C.J., McDougal, J.S., Sowers, L.C., Richman, D.D., and Carson, D.A. 1988. Metabolism and anti‐HIV activity of 2′‐halo‐2′,3′‐dideoxyadenosine derivatives. J. Biol. Chem. 263:5870‐5875.
   Harada, S., Koyanagi, Y., and Yamamoto, N. 1985. Infection of HTLV‐III/LAV in HTLV‐I‐carrying cells MT2 and MT4 and application in a plaque assay. Science (Wash. DC) 229:563‐566.
   Japour, A.J., Mayers, D.L., Johnson, V.A., Kuritzkes, D.R., Beckett, L.A., Arduino, J.M., Lane, J., Black, R.J., Reichelderfer, P.S., D'Aquila, R.T., Crumpacker, C.S., RV‐43 Study group, and the AIDS Clinical Trials Group Virology Committee Resistance Working Group. 1993. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type I isolates. Antimicrob. Agents Chemother. 37:1095‐1101.
   Johnson, V.A., Merrill, D.P., Videler, J.A., Chou, T.‐C., Byington, R.E., Eron, J.J., D'Aquila, R.T., and Hirsch, M.S. 1991. Two‐drug combinations of zidovudine, didanosine, and recombinant interferon‐α inhibit replication of zidovudine‐resistant human immunodeficiency virus type 1 synergistically in vitro. J. Infect. Dis. 164:646‐655.
   Kageyama, S., Weinstein, J.N., Shirasaka, T., Kempf, D.J., Norbeck, D.W., Plattner, J.J., Erickson, J., and Mitsuya, H. 1992. In vitro inhibition of HIV‐1 replication by C2 symmetry‐based HIV protease inhibitors as single agents or in combinations. Antimicrob. Anticancer Chemother. 36:926‐933.
   Koyanagi, Y., Miles, S., Mitsuyasu, R.T., Merrill, J.E., Vinters, H.V., and Chen, I.S. 1987. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science (Wash.DC) 215:819‐822.
   Kusumi, K., Conway, B., Cunningham, S., Berson, A., Evans, C., Iversen, A.K.N., Colvin, D., Gallo, M.V., Coutre, S., Shpaer, E.G., Faulkner, D.V., deRonde, A., Volkman, S., Williams, C., Hirsch, M.S., and Mullins, J.I. 1992. Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro. J. Virol. 66:875‐885.
   Land, S., Treloar, T., McPhee, D., Birch, C., Doherty, R., Cooper, D., and Gust, I.D. 1990. Decreased in vitro susceptibility to zidovudine of HIV isolates obtained from patients with AIDS. J. Infect. Dis. 161:326‐329.
   Larder, B.A., Darby, G., and Richman, D.D. 1989. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science (Wash. DC) 243:1731‐1734.
   Larder, B.A., Chesebro, B., and Richman, D.D. 1990. Susceptibilities of zidovudine‐susceptible and ‐resistant human immunodeficiency virus isolates to antiviral agents determined by using a quantitative plaque reduction assay. Antimicrob. Agents Chemother. 34:436.
   Looney, D.J., Hayashi, S., Nicklas, M., Redfield, R.R., Broder, S., Wong‐Staal, F., and Mitsuya, H. 1990. Differences in the interaction of HIV‐1 and HIV‐2 with soluble CD4. J. Acquired Immune Defic. Syndr. 3:649‐657.
   Mitsuya, H. and Broder, S. 1986. Inhibition of the in vitro infectivity and cytopathic effect of human T‐lymphotropic virus type III/lymphadenopathy associated virus (HTLV‐III/LAV) by 2′,3′‐ dideoxynucleosides. Proc. Natl. Acad. Sci. U.S.A. 83:1911‐1915.
   Mitsuya, H., Matsushita, S., Harper, M.E., and Broder, S. 1985a. Pharmacologic inhibition of infectivity of HTLV‐III in vitro. Cancer Res. 45:4583s‐4587s.
   Mitsuya, H., Weinhold, K.J., Furman, F.A., St. Clair, M.H., Lehrman, S.N., Gallo, R.C., Bolognesi, D., Barry, D.W., and Broder, S. 1985b. 3′‐Azido‐3′‐deoxythymidine (BW A509U): An antiviral agent that inhibits the infectivity and cytopathic effect of human T‐lymphotropic virus type III/lymphadenopathy‐associated virus in vitro. Proc. Natl. Acad. Sci. U.S.A. 82:7096‐7100.
   Mitsuya, H., Matsukura, M., and Broder, S. 1987a. Rapid in vitro systems for assessing activity of agents against HTLV‐III/LAV. In AIDS: Modern Concepts and Therapeutic Challenges (S. Broder, ed.) pp. 303‐333. Marcel Dekker Inc. New York.
   Mitsuya, H., Jarrett, R.F., Matsukura, M., Reitz, M.S., Johns, D.G., and Broder, S. 1987b. Long‐term inhibition of HTLV‐III/LAV DNA synthesis and RNA expression in T cells protected by 2′, 3′‐dideoxynucleosides. Proc. Natl. Acad. Sci. U.S.A. 84:2033‐2037.
   Mitsuya, H., Dahlberg, J.E., Spigelman, Z., Matsushita, S., Jarrett, R.F., Matsukura, M., Currens, M.J., Aaronson, S.A., Reitz, M.S., McCaffrey, R.S., and Broder, S. 1988a. 2′, 3′‐dideoxynucleosides: Broad spectrum antiretroviral activity and mechanism of action. In Human Retrovirus, Cancer, and AIDS: Approaches to Prevention and Therapy, (D. Bolognesi, ed.), pp. 407‐421. Alan R. Liss, New York.
   Mitsuya, H., Looney, D.J., Kuno, S., Ueno, R., Wong‐Staal, F., and Broder, S. 1988b. Dextran sulfate suppression of viruses in the HIV family: Inhibition of virion binding to CD4+ cells. Science (Wash. DC) 240:646‐649.
   Mitsuya, H., Yarchoan, R., and Broder, S. 1990. Molecular targets for AIDS therapy. Science (Wash. DC) 249:1533‐1544.
   Nunberg, J.H., Schleif, W.A., Boots, E.J., O'Brien, J.A., Quintero, J.C., Hoffman, J.M., Emini, E.A., and Goldman, M.E. 1991. Viral resistance to human immunodeficiency virus type 1‐specific pyridinone reverse transcriptase inhibitors. J. Virol. 65:4887‐4892.
   Pauwels, R., Balzarini, J., Baba, M., Snoeck, D., Schols, R., Schols, D., Herdewijn, P., Desmyter, J., and De Clercq, E. 1988. Rapid and automated tetrazolium‐based colorimetric assay for the detection of anti‐HIV compounds. J. Virol. Methods 20:309‐321.
   Richman, D.D. 1990. Zidovudine resistance of human immunodeficiency virus. Rev. Infect. Dis. 12:S507‐S512.
   Richman, D.D., Grimes, J.M., and Lagakos, S.W. 1990. Effect of stage of disease and drug dose on zidovudine susceptibilities of isolates of human immunodeficiency virus. J. AIDS 3:743‐746.
   Richman, D.D., Guatelli, J.C., Grimes, J., Tsiatis, A., and Gingeras, T.R. 1991a. Detection of mutations associated with zidovudine resistance in human immunodeficiency virus utilizing the polymerase chain reaction. J. Infect. Dis. 164:1075‐1081.
   Richman, D., Shih, C.‐K., Lowy, I., Rose, J., Prodanovich, P., Goff, S., and Griffin, J. 1991b. HIV‐1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc. Natl. Acad. Sci. U.S.A. 88:11241‐11245.
   Shirasaka, T., Murakami, K., Ford, H., Kelley, J., Yoshioka, H., Kojima, E., Aoki, S., Driscoll, J.S., Broder, S., and Mitsuya, H. 1990. Halogenated congeners of 2′, 3′‐dideoxypurine nucleosides active against HIV in vitro: A new class of lipophilic prodrugs. Proc. Natl. Acad. Sci. U.S.A. 87:9426‐9430.
   Shirasaka, T., Yarchoan, R., O'Brien, M.C., Husson, R., Anderson, B., Kojima, E., Broder, S., and Mitsuya, H. 1993. Changes in drug‐sensitivity of human immunodeficiency virus type 1 during therapy with azidothymidine, dideoxycytidine, or dideoxyinosine: An in vitro comparative study. Proc. Natl. Acad. Sci. U.S.A. 90:562‐566.
   St. Clair, M.H., Martin, J.L., Tudor‐Williams, G., Bach, M.C., Vavro, C.L., King, D.M., Kellam, P., Kemp, S.D., and Larder, B.A. 1991. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV‐1 reverse transcriptase. Science (Wash. DC) 253:1557‐1559.
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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