In Vitro Evaluations

Antiviral Testing Capabilities

• Evaluations in established human cell lines. These anti-HIV assays are conducted using a wide range of established cell lines including: those of T-cell lineage (CEM-SS, MT-2, MT-4, H9, Jurkat, Molt-4, SupT1, and others); B-cell lineage (AA5, AA2, and others); monocyte-macrophage lineage (U937, THP1, HL60, and others); and other human cell lines (HeLa-CD4-LTR-βgal, MAGI-CCR5, 174xCEM, and others). Anti-HIV assays are routinely performed using a variety of endpoints including:
• Cytoprotection - inhibition of viral cytopathic effects is measured using the tetrazollium dye MTS (Promega) to evaluate cell viability following virus infection.
• Reporter gene induction - cell lines engineered to express reporter genes (e.g., β-galactosidase or luciferase) following HIV infection are used to evaluate antiviral activity.
• Reverse transcriptase (RT) - inhibition of virus replication determined as a reduction in supernatant RT activity.
• ELISA - supernatant or cell-associated HIV-1 p24 antigen content assessed using commercially available ELISA kits.
• Virus infectivity - supernatants from infected cultures are titrated to quantify the relative titer of infectious virus released from the cells.
• Evaluations in primary human cells. Assays are performed using fresh human peripheral blood mononuclear cells (PBMCs) or monocytes-derived-macrophages (MDMs) infected with laboratory or low-passage clinical isolates of HIV-1, HIV-2, or SIV. Blood is purchased commercially (screened for HIV and HBV), and PBMCs are isolated by Ficoll-hypaque centrifugation. The MDMs are isolated by plastic adherence separation from the banded lymphocytes. The PBMCs are stimulated with phytohemagglutin in (PHA) and IL-2 prior to use in the antiviral assays. Antiviral activity is determined as a reduction in supernatant reverse transcriptase activity and/or p24 antigen content 6 days post-infection.
• Virus isolates. More than 200 HIV-1 isolates (lab-adapted and clinical; CXCR4-, CCR5-, and Dual-tropic; drug-sensitive and drug-resistant) are available for antiretroviral evaluations. These include isolates from HIV-1 Group M subtypes A, B, C, D, E, F, and G, Group O, and Group N. Virus panels include HIV-1 drug-resistant isolates (NRTI-, NNRTI-, PI-, INI-, and multidrug resistant), as well as HIV-2 and SIV isolates.
• Antiviral activity in the presence of serum proteins and inhibitory quotient
• Cytoxicity and therapeutic indices. Evaluating the cytotoxicity of a test compound is an important aspect of understanding the specificity of the compound's antiviral activity. Therefore, all antiviral assays conducted at Southern Research include a parallel cytotoxicity assay to allow for the determination of a compound's in vitro therapeutic index, which is the ratio of the compound's 50% toxic concentration to the compound's 50% inhibitory concentration against virus replication (TI=TC₅₀/IC₅₀).
• In vitro combination activity. Combination drug therapy is the current paradigm for clinical treatment of HIV infection. As such, it is critical to evaluate the activity of developmental test compounds in combination with clinically relevant agents being used in the treatment of HIV-infected patients. A panel of all currently FDA-approved antiretroviral drugs, as well as other clinically relevant drugs, is available for two-drug combination studies, which can be performed using any of the in vitro anti-HIV assays available at Southern Research. Data are evaluated using the Prichard and Shipman MacSyngery II software; however, other methods, such as Chou and Talalay median dose effect equation as well as ComboSTAT, can be used upon request. The interactions of the compounds are evaluated in terms of antiviral efficacy and cytotoxicity to identify synergistic, additive, or antagonistic interactions.
• Resistance. The in vitro selection of virus that is resistant to the antiviral activity of a test compound is performed to determine the genetic barrier to resistance for a developmental drug (i.e., how quickly resistance might emerge during treatment), as well as to confirm the drug's mechanism of action (i.e., for direct-acting antivirals, mutations are expected to emerge in the viral gene for the drug's target protein). Dose escalation and single high-dose models are used for selection of resistant viruses. Isolated resistant viruses are evaluated phenotypically to determine the fold resistance compared to wild-type virus as well as genotypically to identify resistance-associated mutations that emerge in the viral genome. Generation of HIV molecular clones containing the identified mutations also can be performed to confirm the association of specific mutations with resistance.
• Other anti-HIV and mechanism-based assays. A variety of other anti-HIV assays are performed in our laboratory to address special requirements of the sponsor or compound, including:
• Assays for evaluating HIV entry inhibitors or topical microbicides. Compounds may be evaluated for inhibitory activity in CD4-dependent and CD4-independent HIV transmission assays. CXCR4- and CCR5-tropic systems are available for evaluating compounds in the assays listed below.
• Attachment assays. MAGI cells are infected with virus in the presence of test compound. Compound and unbound virus are washed out after two hours. Inhibition of virus attachment is determined as a reduction in virus-induced β-galactosidase enzyme expression using a chemiluminescence detection method after 48-hour incubation.
• Fusion assays. Co-cultivation of MAGI cells with HIV-1 Env and Tat expressing cells results in CD4-dependent HIV-1 Env mediated cell fusion, which leads to cytoplasmic mixing of the contents of the two cell lines and subsequent Tat transactivation of an HIV-1 LTR β-galactosidase reporter construct. Inhibition of cell-cell fusion is determined as a reduction in β-galactosidase enzyme expression using a chemiluminescence detection method after 48-hour incubation.
• Cell-cell transmission assays. Adherent target cells are co-cultivated for four hours with a chronically infected suspension cell line in the presence of test compound. Inhibition of cell-to-cell virus transmission is determined by analysis of cell-associated p24 after 24-hour incubation (CD4-dependent assays) or by analysis of supernatant p24 after six-day incubation (CD4-dependent assay).
• Seminal plasma and pH transition assays. Attachment or cell-cell transmission assays are performed in the presence of human seminal plasma or pH transition from 4.0 to 7.0, which mimics the pH change that occurs during sexual intercourse.
• Virucidal assays. Compounds are evaluated for their ability to directly inactivate virus. Compounds can be evaluated for direct or indirect virucidal activity in cell-free and cell-associated models. Virus is incubated with test compound (one or multiple concentrations) for various periods of time. Drug is removed by centrifugation and washing of the resulting virus pellet. Virus inactivation can be determined using a variety of endpoint assays (e.g., infection of MAGI cells).
• Lactobacillus toxicity assay. The rate of HIV-1 transmission has been correlated with the presence or absence of beneficial H₂O₂-secreting Lactobacilli species in the vaginal vault. Lack of toxicity to these beneficial bacteria of the vagina is expected of a topical microbicide candidate. Inhibition of bacterial growth for two common Lactobacilli sp. (L. jensenii and L. crispatus) is determined by measurement of optical density at 490 nm following 24-hour incubation with test compound.
• Cervical explant assay. In this acute infection assay using fresh human cervical tissues, antiviral activity is determined by a reduction in supernatant p24 over a 14-day culture period compared to an untreated virus control. Compounds are tested at three concentrations. Tissue toxicity can also be determined in a parallel MTT-based assay.
• Chronic and latent infection assays. Compounds are evaluated for their ability to inhibit virus production from cells (i.e., CEM-SS, H9, MT-2, etc.) chronically infected with a variety of laboratory adapted viruses, or from latently infected cell lines (e.g., U1 and ACH-2) induced to produce virus in response to stimulation by phorbol esters or tumor necrosis factor alpha. In addition to cell lines, we also have the capability to analyze the effect of compounds on PBMCs chronically infected with a variety of low-passage HIV-1 virus isolates.
• HIV-1 Tat and Rev inhibition assays. Compounds are evaluated in engineered HeLa cell lines with either HIV-1 Tat-dependent or Rev-dependent expression of Renilla Luciferase (RLuc). Inhibition of Tat or Rev function is determined as a reduction in RLuc expression. Nonspecific compound activity (e.g., cytotoxicity) is measured in parallel as a reduction in the Tat- or Rev-independent expression of Firefly Luciferase in the same assay well.
• Time of addition and pre-/post-treatment variation assays. Evaluations are performed to determine the activity of a compound when present only before infection, only after infection, or when added to cultures at various times pre-and post-infection to determine the general point in the virus life cycle inhibited by the compound. Relevant control drugs (e.g, entry, RT, and integrase inhibitors) are included as markers for specific viral targets. These assays can be performed at low or high MOI and may also include multiple addition of the compound over extended periods of time.
• Target-based biochemical assays. Commercially available kit-based biochemical assays for HIV-1 enzymes (Protease, Reverse Transcriptase, Integrase) are used to evaluate compounds for inhibition of these targets. Fluorescence anisotropy (polarization) assays for HIV-1 Tat/TAR and Rev/RRE binding are also available.

Vaccine Testing Capabilities

For information about our vaccine testing capabilities, visit Vaccine Development page.

Animal Model Support Assays

For information about our animal support assays, visit our Vaccine Development page.

Custom Assay Development

In addition to our comprehensive range of standardized assays, we also offer clients the capability to develop customized assays to meet their specific antiretroviral research needs.