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79 A Rapid Multi-Functional HIV-1 Entry Assay for Measuring Neutralizing Antibody, Coreceptor Tropism, and Drug Susceptibility
T. Wrin, W. Huang, J. Yap, S. Fransen, J. Beauchaine, N. Whithurst, M. Reddy, S. Nidtha, E. E. Paxinos, N. T. Parkin, J. W. Whitcomb, and C. J. Petropoulos*
ViroLogic, Inc., South San Francisco, CA, USA
Neutralizing antibodies and drug inhibitors of HIV-1 entry interfere with infection of cells by HIV-1 by perturbing the early steps in viral entry. We are using recombinant viruses in a single replication cycle assay to explore the interactions of envelope gp120SU and gp41TM with the cellular receptors (CD4, CCR5, and CXCR4) and to assess the effects of antibodies and entry inhibitors on infection. The assay is performed by: (a) generating HIV-1 particles that carry a firefly luciferase gene and are pseudotyped with patient virus encoded envelope proteins, (b) infecting cells expressing CD4 plus CCR5 and/or CXCR4, and (c) measuring luciferase production resulting from a single round of virus replication in the presence and absence of inhibitors or neutralizing antibodies. R5-, X4-, CD4-, and fusion inhibitors have been successfully evaluated using the assay for both laboratory- and patient-derived envelopes. Patient plasmas have been evaluated for the presence of neutralizing antibody against a variety of laboratory and primary strains. IC50s determined for drugs and antibodies in the different cell types vary and may be related to the relative receptor levels on the cell surface. In general, cell types with higher cell surface receptor levels yield higher IC50s. In a given cell line, the IC50s of R5-, X4-, CD4-, and fusion inhibitors vary up to 40-fold among drug naïve primary viruses. Plasmas from HIV-1-infected people yield a broad range of neutralization titers (<1:10 to 1:20,000) against primary viruses of various cell tropisms. This study demonstrates that susceptibility to neutralizing antibodies and virus entry inhibitors as well as determination of X4/R5 coreceptor tropism are amenable to evaluation with recombinant virus assays. Screening of patient plasma for neutralizing antibody against a standard panel of viruses is completed in 3 days. This assay is a rapid and convenient way to measure antibody neutralization elicited by HIV vaccine candidates.
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