title: A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.
abstract: Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4+T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4+T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Axel
authors.initials: A
authors.lastname: Fun
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Hoi Ping
authors.initials: HP
authors.lastname: Mok
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wills
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lever
mesh_terms: D000328:Adult; D000368:Aged; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002469:Cell Separation; D002478:Cells, Cultured; D005260:Female; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D019562:Viral Load; D017735:Virus Latency; D014779:Virus Replication