title: Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling.
abstract: Cultured primary peripheral blood mononuclear cells (PBMC) represent a potentially physiologic in vitro model of HIV-1 infection, but assessment of antibody-mediated HIV-1 neutralization using PBMC has been hindered by donor variability and lack of a sustainable individual PBMC source. To advance this model for HIV vaccine evaluation, intra- and inter-assay variability were assessed using monoclonal and polyclonal antibodies and PBMC targets from multiple HIV-seronegative donors. Inter-assay variability was introduced by using different PBMC for virus propagation, and more substantially, for assay targets. Neutralization titers varied by as much as 4 logs when using different individual donor PBMC as targets; variability was antibody-specific, with the greatest variation observed using an individual polyclonal plasma. Pooling of multiple PBMC donors significantly reduced median inter-assay variation to the level of intra-assay variation, suggesting a pathway forward for establishing a uniform, sustainable and standardized approach to the assessment of antibody function using a PBMC model.
authors.affiliation: Military HIV Research Program, Silver Spring, MD, USA; Henry M. Jackson Foundation, Bethesda, MD, USA.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Wieczorek
authors.forename: Bruce K
authors.initials: BK
authors.lastname: Brown
authors.forename: Camila
authors.initials: C
authors.lastname: Delsarto Macedo
authors.forename: Maggie
authors.initials: M
authors.lastname: Wesberry-Schmierer
authors.forename: Viseth
authors.initials: V
authors.lastname: Ngauy
authors.forename: Andrew
authors.initials: A
authors.lastname: Rosa Borges
authors.forename: Nelson L
authors.initials: NL
authors.lastname: Michael
authors.forename: Mary A
authors.initials: MA
authors.lastname: Marovich
authors.forename: David C
authors.initials: DC
authors.lastname: Montefiori
authors.forename: Victoria R
authors.initials: VR
authors.lastname: Polonis
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology*; D002478:Cells, Cultured; D015483:HIV Antibodies / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D009500:Neutralization Tests / Q000379:methods*; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*
chemical_list: D057134:Antibodies, Neutralizing; D015483:HIV Antibodies
keywords: HIV-1 neutralization; Infectious molecular clones; Monoclonal antibodies; PBMC targets; Peripheral blood mononuclear cells; Primary isolates; Standardization