@article{ffc4ce8ab548443aa0aff1806661319f,
title = "Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC",
abstract = "Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.",
keywords = "Assay standardization, Envelope glycoprotein, HIV neutralization, HIV reporter virus, HIV-1, Luciferase, Neutralizing antibody, PBMC assay, Vaccine assessment",
author = "Edmonds, {Tara G.} and Haitao Ding and Xing Yuan and Qing Wei and Smith, {Kendra S.} and Conway, {Joan A.} and Lindsay Wieczorek and Bruce Brown and Victoria Polonis and West, {John T.} and Montefiori, {David C.} and Kappes, {John C.} and Christina Ochsenbauer",
note = "Funding Information: This work was supported by the NIH Center for HIV/AIDS Vaccine Immunology (CHAVI) , UO1-AI067854 ; the Bill & Melinda Gates Foundation's Collaboration for AIDS Vaccine Discovery (CAVD)/Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC) , grant number 38619 ; facilities of the Virology and Genetic Sequencing cores of the UAB Center for AIDS Research (P30-AI-27767); and the Genetically Defined Microbe and Expression Core of the UAB Mucosal HIV and Immunobiology Center (R24 DK-64400). Research was also supported by a Merit Review Award (JCK), from the Department of Veterans Affairs Medical Center, Research Services . TGE is supported by NIH training grant AI007439 . We would like to acknowledge the expertise and support we obtained from the Statistical Center for HIV/AIDS Research and Prevention (SCHARP), Fred Hutchinson Cancer Research Center, Seattle, WA. Funding Information: Human primary peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient from buffy coats obtained from healthy HIV-1 seronegative donors. Buffy coats were purchased from Research Blood Components (Brighton, MA) or purified PBMC were provided by Tom Denny (Duke University) and the CAVD CTC-VIMC, as funded by the Bill & Melinda Gates Foundation (grant number 38650). PBMC were cultured in RPMI 1640 growth medium supplemented with penicillin (100 U/mL), streptomycin (100 U/mL), L-glutamine (2 mM), interleukin-2 (IL-2) (30 U/mL) (Roche, Indianapolis, IN) and 10% FBS. The cells were stimulated by culturing in growth medium containing phytohemagglutinin (PHA)-P (2–5 μg/mL) (Sigma-Aldrich) for 24 h. The next day, the medium was removed and the cells were placed into culture with RPMI 1640 containing IL-2 (30 U/mL) and used either immediately, or within 4 days. ",
year = "2010",
month = dec,
day = "5",
doi = "10.1016/j.virol.2010.08.028",
language = "English (US)",
volume = "408",
pages = "1--13",
journal = "Virology",
issn = "0042-6822",
publisher = "Academic Press Inc.",
number = "1",
}