title: Comparison of immunologic assays for detecting immune responses in HIV immunotherapeutic studies: AIDS Clinical Trials Group Trial A5181.
abstract: 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.
authors.affiliation: Division of Infectious Diseases/HIV/AIDS Unit, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. macatangaybj@upmc.edu
authors.forename: Bernard J C
authors.initials: BJ
authors.lastname: Macatangay
authors.forename: Lu
authors.initials: L
authors.lastname: Zheng
authors.forename: Charles R
authors.initials: CR
authors.lastname: Rinaldo
authors.forename: Alan L
authors.initials: AL
authors.lastname: Landay
authors.forename: Richard B
authors.initials: RB
authors.lastname: Pollard
authors.forename: Savita
authors.initials: S
authors.lastname: Pahwa
authors.forename: Michael M
authors.initials: MM
authors.lastname: Lederman
authors.forename: R Pat
authors.initials: RP
authors.lastname: Bucy
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D019380:Anti-HIV Agents / Q000627:therapeutic use; D000956:Antigens, Viral; D023241:Antiretroviral Therapy, Highly Active / Q000379:methods; D049109:Cell Proliferation; D005260:Female; D005434:Flow Cytometry; D015658:HIV Infections / Q000188:drug therapy / Q000276:immunology* / Q000628:therapy*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007167:Immunotherapy / Q000379:methods*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000378:metabolism; D007376:Interleukin-2 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008297:Male; D008875:Middle Aged; D014773:Virology / Q000379:methods*; D055815:Young Adult
chemical_list: D019380:Anti-HIV Agents; D000956:Antigens, Viral; D007376:Interleukin-2; D007371:Interferon-gamma