High-throughput Quantitative Analysis of HIV-1 and SIV-specific ADCC-mediating Antibody Responses

Cytometry A. 2011 Aug;79(8):603-12. doi: 10.1002/cyto.a.21084. Epub 2011 Jul 6.

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.

MeSH terms

  • AIDS Vaccines / immunology*
  • Animals
  • Antibodies, Viral / analysis*
  • Antibody-Dependent Cell Cytotoxicity*
  • Flow Cytometry / methods
  • Granzymes / immunology
  • HIV Antibodies / analysis*
  • HIV Infections / prevention & control
  • HIV-1 / immunology*
  • Haplorhini
  • High-Throughput Screening Assays*
  • Humans
  • Killer Cells, Natural / cytology
  • Killer Cells, Natural / immunology
  • Receptors, IgG / immunology
  • SAIDS Vaccines / immunology*
  • Simian Immunodeficiency Virus / immunology*
  • Vaccination

Substances

  • AIDS Vaccines
  • Antibodies, Viral
  • HIV Antibodies
  • Receptors, IgG
  • SAIDS Vaccines
  • Granzymes