Infectious Disease Research

Virology
Retroviruses

Anti-HIV Evaluation Assays

1. Evaluations in established human cells
2. Evaluations in mechanism-based assays
3. Evaluations in fresh human cells
4. Topical Microbicide Assays For Inhibitors of Sexual Transmission
5. Other Anti-HIV Assays

• Evaluations in established human cells: An established, high capacity anti-HIV drug screening program is available for the primary analysis of compounds in a microtiter assay which measures the ability of selected compounds to inhibit HIV-induced cell killing as well as the toxicity of the test compounds to host cells. Quantitation is performed spectrophotometrically using the tetrazolium dye MTS (Cell Titer; Promgega) which is converted to a soluble, colored formazan product by mitochondrial enzymes present in metabolically active cells at six days post-infection. Confirmatory assays include reverse transcriptase, p24 and infectious virus assays, as well as macroscopic and microscopic observation of test wells. Additionally, this assay may be tailored to the clients needs by inclusion of alternative measurements of cell survival, such as 3H metabolite incorporation or assessment by alternative dye indicators or formulations. This assay system has been used to perform over 25,000 dose-response tests per year. The basic assay involves infection of CEM-SS cells with virus in the presence of the test compound. Data are analyzed using a statistical software program developed at Southern and efficacy and toxicity endpoints are determined, as well as selectivity indices.

  EFFICACY AND TOXICITY OF REPRESENTATIVE ANTI-HIV COMPOUNDS IN XTT-BASED ASSAY

  Compound	Mechanism of Action	IC 50 (m M)	TC 50 (m M)
  ISIS 5320	A/F	0.5	125
  CSB	A/F	9.5 ug/ml	46.9
  Cosalane	A/F	4.3 ug/ml	>100 ug/ml
  dextran sulfate	A/F	1.6 ug/ml	>100 ug/ml
  AZT	NRT	0.001	32.0
  ddC	NRT	0.01	>1.0
  Ddl	NRT	1.8 ug/ml	>100 ug/ml
  d4T	NRT	0.3	>100
  Carbovir	NRT	0.4	>2.0
  TIBO	NNRTI	0.07	>10
  DPS	NNRTI	1.9	>30
  Thiazolobenzimidazole	NNRTI	0.4	13.6
  Benzothiadiazines	NNRTI	1.4	>20
  Nevirapine	NNRTI	0.11	>10
  KNI 272	P	0.5	>2.0
  Castanospermine	G	72.2 ug/ml	>100 ug/ml
  N-butyl-DNJ	G	100	>400
  Hydantoin	SURFACE	27	>267

  
  Primary anti-HIV assays are routinely performed in CEM-SS, MT2, or MT4 cells infected with either the IIIB or RF strains of HIV-1. We also perform anti-HIV assays in a variety of other established human cell lines, including those of the T-cell lineage (H9, Jurkat, Molt-4, SupT1), B-cell lineage (AA5, AA2) and monocyte-macrophage lineage (U937, THP1, HL60), as well as other human cell lines (HeLa-CD4, HeLa-CD4-LTR-b -galactosidase, 174xCEM, etc.). Endpoints in these assays may involve quantitation of virus growth such as reverse transcriptase, p24 and/or infectious virus (see Confirmatory Anti-HIV Assays) due to variations in virus cytopathogeneicity in the various cell lines. Antiviral assays may be performed using a wide variety of laboratory virus isolates as well as primary isolates with the ability to grow in established cell lines such as MT2 cells. These isolates include IIIB, MN, RF, LAV, SF2, NL4-3 as well as a panel of laboratory isolates obtained from patients.
  
  
• Evaluations in mechanism-based assays: In addition to the cell-based assays described above, high-throughput, mechanism-based assays are performed to evaluate the ability of compounds to inhibit gp120-CD4 interaction, reverse transcriptase, RNaseH, Integrase, Protease, Tat, Rev and Nef. These biochemical assays, as well as their cell-based counterparts, are described below (see Cell-Based and Biochemical Mechanism of Action). For high-throughput evaluation, the assays may be performed robotically (see High-Throughput Screening and Target Development).
  
  
• Evaluations in fresh human cells: Evaluations are performed in fresh human peripheral blood lymphocytes and monocyte-macrophages infected with low passage primary virus isolates. Blood is obtained from the American Red Cross (screened for HIV and HBV) and mononuclear cells are isolated by Ficoll-hypaque centrifugation. Monocyte-macrophages are isolated by adherence separation from the banded lymphocytes or obtained commercially as elutriated cells. Leukocytes are stimulated with phytohemagglutinin (PHA) or IL2 for use in the antiviral assays. Clinical virus isolates have been obtained by cocultivation with patient lymphocytes. Isolates representative of each of the virus clades found worldwide are available for use. The available isolates have been characterized for (a) sensitivity to AZT, ddI, ddC and nevirapine, (b) syncytium-inducing (SI) versus non-syncytium-inducing (NSI) phenotype, (c) growth potential in PBMCs (i.e., slow/low versus rapid/high) and (d) tropism (lymphotropic, macrophage-tropic). These virus isolates are passaged minimally in fresh cells and never in established human cells. Assay results are quantitated by reverse transcriptase, p24 or infectious virus assays (see Confirmatory Anti-HIV Assays).
  

  BIOLOGICAL PROPERTIES OF CLINICAL STRAINS OF HIV-1

  ISOLATE	TROPISM	AZT IC50 (mM)	ddI IC50 (mM)	SYNCYTIA	GROWTH
  BAKI	LYMPHOCYTE	0.049	2.61	SI	RAPID/HIGH
  SLKA	MACROPHAGE	0.025	0.32	NSI	SLOW/LOW
  WEJO	LYMPHOCYTE	0.056	2.18	SI	RAPID/HIGH
  ROJO	LYMPHOCYTE	0.016	0.87	SI	RAPID/HIGH
  ROMA	MACROPHAGE	0.016	0.16	SI	RAPID/HIGH
  STDA	LYMPHOCYTE	0.017	0.23	SI	RAPID/HIGH
  WOME	LYMPHOCYTE	0.016	0.41	SI	RAPID/HIGHT
  VIHU	LYMPHOCYTE	0.016	1.21	NSI	SLOW/LOW
  TEKI	LYMPHOCYTE	0.029	0.37	NSI	SLOW/LOW
  TEKI	LYMPHOCYTE	0.016	1.70	NSI	SLOW/LOW
  DEJO	LYMPHOCYTE	0.015	ND	NSI	SLOW/LOW
  BLCH	LYMPHOCYTE	0.010	ND	NSI	SLOW/Low
  RIARL	LYMPHOCYTE	0.010	ND	NSI	SLOW/LOW

  
  
• Topical Microbicide Assays For Inhibitors of Sexual Transmission: Compounds may be evaluated for inhibitory activity in CD4 dependent and CD4 independent transmission assays. Target cells expressing HIV-1 coreceptors with (GHOST X4/R5) or without (ME180, cervical epithelial cells) CD4 can be infected by cocultivation with HIV-infected lymphocytes. Endpoint quantitation is performed by p24 ELISA (efficacy) and XTT (toxicity). Compounds may also be evaluated in the presence of mucin to simulate the protein enriched secretions present in the vaginal and anal vaults. Secondary evaluations include specific mechanism of action assays to identify virucidal compounds, attachment, fusion and reverse transcriptase inhibitors. Additionally, compounds may be evaluated for toxicity to normal vaginal flora, i.e., lactobacillus sp.
  
  
• Other Anti-HIV Assays: A variety of other anti-HIV assays are performed in our laboratory to address special requirements of the sponsor or compound. These include:
  • Syncytial inhibition assays in CEM-SS, HeLa-CD4 or HeLa-CD4-LTR-b -galactosidase cells with immunofluorescent chemiluminescence or colorimetric endpoint.
  • Attachment and fusion inhibition assays using indicator cell lines and quantitation by chemiluminescent, colorimetric or microscopic evaluation.
  • Latent inhibition assays using established cell lines (U1, ACH-2, OM10.1) persistently infected with HIV and using a p24 or RT endpoint.
  • Chronically infected cell inhibition assay using either chronically infected HIV-1 Peripheral blood mononuclear cells or cell lines. Chronically infected cell inhibition assay using either chronically infected HIV-1 Peripheral blood mononuclear cells or cell lines.

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Anti-HIV Evaluation
Confirmatory HIV Assays
Secondary Biological Assays
Topical Microbicide Program
Combination Assays

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