ChemDiv staff offers decades of experience in the HIV research and drug development. Multiple cell-based assays to evaluate anti-HIV screening compounds are available, including replication assays with primary (PBMC cells) or transformed T cell lines, and use of reporter T cell lines to monitor virus infectivity. In parallel with antiviral assays, our services include evaluation of compound’s cytotoxic effects in several uninfected cell lines. We utilize a variety of reporter genes and readouts adapted to the specific needs of each client’s assays. In addition, we offer a number of customized assays to investigate the mechanism of action of drug candidates, such as the emergence of resistant strains and characterization of viral targets. We also perform screening of small molecule libraries with several cell-based assays developed in-house or following the client’s needs. Known HIV inhibitors are being used as positive control in all the assays.
Anti-HIV efficacy in primary cells and T cell lines
Cell-based antiviral assays are performed with either transformed T cell lines (SupT1, H9, Molt4), primary blood-derived mononuclear cells (PBMCs) or macrophages. PBMC cells are prepared, stimulated and then infected with HIV laboratory-adapted or primary isolates utilizing different coreceptors. Experimental readout of HIV replication can be performed by ELISA of p24 viral antigen, monitoring reverse transcriptase, reporter virus or by intracellular or surface staining of viral antigens (e.g. Gag protein or Env, respectively). Antiviral assays can be performed with a set of primary and laboratory adapted HIV isolates (displaying X4, R5 or dual tropism), and virus resistant to each family of ARVs. Multi-drug resistant isolates are also available. In vitro antiviral assays can be performed at low and medium throughputs (up to 100,000 molecules).
Single-cycle infectivity assays
A variety of stable reporter cell lines expressing HIV receptor and coreceptors are used to evaluate infectivity of HIV or HIV-based pseudotypes using single cycle assays. We also offer multi-tiered assays to evaluate the antiviral activity of late-stage antiviral screening compounds (e.g. maturation inhibitors). These latter assays require production of viral particles in compound treated cells and subsequent analysis of infectivity in permissive cells in the absence of a screening compound.
Mechanism of action studies
“time of addition” assays in synchronized infections
evaluation of viral entry inhibition
determination of HIV reverse transcriptase activity
Determination of HIV integrase activity
Resistance Selection and Characterization
Resistant selection may be carried out in either transformed or primary cells with a variety of viral isolates. Selections for multidrug resistant variants are available using a combination of antiviral compounds including approved drugs likely to be used in combination therapy in the clinic. Characterization of resistant variants includes determination of genotypic and phenotypic variations.
Drug combination studies
Combination studies are performed with multiple classes of drugs including the following:
nucleoside reverse transcriptase inhibitors
non-nucleoside reverse transcriptase inhibitors
Efficacy of combination antivirals is evaluated as synergistic, additive or antagonistic using software packages including Prichard and Shipman MacSynergy II and Chou and Talalay median dose effect (Calcusyn).
Pseudovirions to evaluate viral entry inhibitors
Pseudotyping HIV virus with heterologous viral envelope proteins can be used as a surrogate assay to evaluate viral entry in a number of viruses. These studies have been used to identify and study candidate viral entry inhibitors against HCV, Dengue virus, West Nile virus, Ebola, Yellow Fever, highly pathogenic avian influenza and many other infectious agents for which entry assays are difficult to perform because they are not available, or when available, they require the use of highly infectious virus under strict Biosafety level conditions (BSL3 or BSL4).
Additional HIV assays
Other available assays include evaluation of HIV protease activity, entry assays, integration assays, antibody neutralization assays, coreceptor determination, HIV-induced downmodulation of CD4 and class I MHC, anti-HIV activity in chronically infected cells (with established HIV replication), evaluation of CPE effect in infected cells (syncytia formation, apoptosis, direct killing). These assays will help address the mechanism of action of lead screening compounds. ChemDiv also offers customized assays adapted to the specific needs of our clients. Additional information on these and other assays are available upon request. Our staff will work with you to develop additional assays appropriate to your needs.