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High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.

Publication ,  Journal Article
Pollara, J; Hart, L; Brewer, F; Pickeral, J; Packard, BZ; Hoxie, JA; Komoriya, A; Ochsenbauer, C; Kappes, JC; Roederer, M; Huang, Y ...
Published in: Cytometry A
August 2011

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.

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Published In

Cytometry A

DOI

EISSN

1552-4930

Publication Date

August 2011

Volume

79

Issue

8

Start / End Page

603 / 612

Location

United States

Related Subject Headings

  • Vaccination
  • Simian immunodeficiency virus
  • Simian Immunodeficiency Virus
  • SAIDS Vaccines
  • Receptors, IgG
  • Killer Cells, Natural
  • Immunology
  • Humans
  • High-Throughput Screening Assays
  • Haplorhini
 

Citation

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Pollara, J., Hart, L., Brewer, F., Pickeral, J., Packard, B. Z., Hoxie, J. A., … Ferrari, G. (2011). High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A, 79(8), 603–612. https://doi.org/10.1002/cyto.a.21084
Pollara, Justin, Lydia Hart, Faraha Brewer, Joy Pickeral, Beverly Z. Packard, James A. Hoxie, Akira Komoriya, et al. “High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.Cytometry A 79, no. 8 (August 2011): 603–12. https://doi.org/10.1002/cyto.a.21084.
Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, et al. High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A. 2011 Aug;79(8):603–12.
Pollara, Justin, et al. “High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.Cytometry A, vol. 79, no. 8, Aug. 2011, pp. 603–12. Pubmed, doi:10.1002/cyto.a.21084.
Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, Komoriya A, Ochsenbauer C, Kappes JC, Roederer M, Huang Y, Weinhold KJ, Tomaras GD, Haynes BF, Montefiori DC, Ferrari G. High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A. 2011 Aug;79(8):603–612.
Journal cover image

Published In

Cytometry A

DOI

EISSN

1552-4930

Publication Date

August 2011

Volume

79

Issue

8

Start / End Page

603 / 612

Location

United States

Related Subject Headings

  • Vaccination
  • Simian immunodeficiency virus
  • Simian Immunodeficiency Virus
  • SAIDS Vaccines
  • Receptors, IgG
  • Killer Cells, Natural
  • Immunology
  • Humans
  • High-Throughput Screening Assays
  • Haplorhini