title: Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.
abstract: A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally.
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC, USA; Department of Surgery, Duke University Medical Center, Durham, NC, USA; Duke Human Vaccine Institute, Durham, NC, USA. Electronic address: marcella.sarzottikelsoe@dm.duke.edu.
authors.forename: Marcella
authors.initials: M
authors.lastname: Sarzotti-Kelsoe
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Xiaoju
authors.initials: X
authors.lastname: Daniell
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Todd
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Miroslawa
authors.initials: M
authors.lastname: Bilska
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Martelli
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Celia
authors.initials: C
authors.lastname: LaBranche
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Lautaro G
authors.initials: LG
authors.lastname: Perez
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Ochsenbauer
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Kappes
authors.affiliation: Duke Human Vaccine Institute, Durham, NC, USA.
authors.forename: Wes
authors.initials: W
authors.lastname: Rountree
authors.affiliation: Duke Human Vaccine Institute, Durham, NC, USA.
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Denny
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Montefiori
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood*; D057205:Automation, Laboratory / Q000592:standards; D015415:Biomarkers / Q000097:blood; D002460:Cell Line; D019983:Guideline Adherence / Q000592:standards; D015483:HIV Antibodies / Q000097:blood*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D057166:High-Throughput Screening Assays / Q000592:standards*; D006801:Humans; D057230:Limit of Detection; D009500:Neutralization Tests / Q000592:standards*; D015588:Observer Variation; D017410:Practice Guidelines as Topic / Q000592:standards; D011237:Predictive Value of Tests; D011786:Quality Control; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection
chemical_list: D057134:Antibodies, Neutralizing; D015415:Biomarkers; D015483:HIV Antibodies
keywords: A3R5 cells; Assay validation; HIV; Neutralizing antibody