Comparison of immunologic assays for detecting immune responses in HIV immunotherapeutic studies: AIDS Clinical Trials Group Trial A5181

Clin Vaccine Immunol. 2010 Sep;17(9):1452-9. doi: 10.1128/CVI.00498-09. Epub 2010 Jul 14.


This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-gamma) cytokine production (CP), single-cell IFN-gamma production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-gamma and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of <3,000 copies/ml (group B). The lymphocyte proliferation assay (LPA) did not significantly differentiate between the two groups. Using fresh peripheral blood mononuclear cells (PBMCs), the CP and ELISPOT assays for IFN-gamma detected the greatest differences between the two groups, specific for three of the five HIV-1 antigens, whereas significant differences were seen only in response to one antigen when cryopreserved cells were used. The strongest correlations were seen between the CP and ELISPOT assays. The ELISPOT B-LCL assay showed a cell concentration-dependent increase in IFN-gamma production compared to that shown by the standard ELISPOT assay but did not differentiate between the groups. In the ICC assay, greater numbers of IFN-gamma-producing T cells were seen in group B, and little or no detectable IL-2 production was seen in both groups. These studies highlight complexities of immunologic monitoring of T-cell responses in multisite clinical trials in HIV infection and outline considerations for optimizing these efforts.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Anti-HIV Agents / therapeutic use
  • Antigens, Viral
  • Antiretroviral Therapy, Highly Active / methods
  • Cell Proliferation
  • Female
  • Flow Cytometry
  • HIV Infections / drug therapy
  • HIV Infections / immunology*
  • HIV Infections / therapy*
  • HIV-1 / immunology*
  • Humans
  • Immunoassay / methods
  • Immunotherapy / methods*
  • Interferon-gamma / biosynthesis
  • Interferon-gamma / metabolism
  • Interleukin-2 / biosynthesis
  • Leukocytes, Mononuclear / immunology
  • Male
  • Middle Aged
  • Virology / methods*
  • Young Adult


  • Anti-HIV Agents
  • Antigens, Viral
  • Interleukin-2
  • Interferon-gamma