title: High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.
abstract: We have developed a high-throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following: (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4(+) T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6 ± 2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.
authors: Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA. justin.pollara@duke.edu
authors: Justin
authors: J
authors: Pollara
authors: Lydia
authors: L
authors: Hart
authors: Faraha
authors: F
authors: Brewer
authors: Joy
authors: J
authors: Pickeral
authors: Beverly Z
authors: BZ
authors: Packard
authors: James A
authors: JA
authors: Hoxie
authors: Akira
authors: A
authors: Komoriya
authors: Christina
authors: C
authors: Ochsenbauer
authors: John C
authors: JC
authors: Kappes
authors: Mario
authors: M
authors: Roederer
authors: Ying
authors: Y
authors: Huang
authors: Kent J
authors: KJ
authors: Weinhold
authors: Georgia D
authors: GD
authors: Tomaras
authors: Barton F
authors: BF
authors: Haynes
authors: David C
authors: DC
authors: Montefiori
authors: Guido
authors: G
authors: Ferrari
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology*; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis*; D000920:Antibody-Dependent Cell Cytotoxicity; D005434:Flow Cytometry / Q000379:methods; D053804:Granzymes / Q000276:immunology; D015483:HIV Antibodies / Q000032:analysis*; D015658:HIV Infections / Q000517:prevention & control; D015497:HIV-1 / Q000276:immunology*; D000882:Haplorhini; D057166:High-Throughput Screening Assays; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology; D018449:SAIDS Vaccines / Q000276:immunology*; D015302:Simian Immunodeficiency Virus / Q000276:immunology*; D014611:Vaccination
chemical_list: D016915:AIDS Vaccines; D000914:Antibodies, Viral; D015483:HIV Antibodies; D017452:Receptors, IgG; D018449:SAIDS Vaccines; D053804:Granzymes